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Respiratory Tract Microbiota Profiling Experiments using OpenArray Application Guide (Pub. No. MAN0017952 Rev. C.0)

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Respiratory Tract Microbiota Profiling Experiments using OpenArray

Respiratory Tract Microbiota Profiling Experiments

C.0 4 January 2021 • Updated instructions to order Custom TaqMan™ OpenArray™ Plates. • Corrected information for TaqMan™ OpenArray™ Respiratory Tract Microbiota Plate (see Table 3 on page 9). Provided additional informa…

Respiratory Tract Microbiota Profiling Experiments - Thermo ...

C.0. 4 January 2021. •. Updated instructions to order Custom TaqMan™ OpenArray™ Plates. •. Corrected information for ...

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Respiratory Tract Microbiota Profiling Experiments
APPLICATION GUIDE TaqManTM Assays for respiratory tract microbiota profiling experiments using OpenArrayTM plates
for use with: TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate Custom TaqManTM OpenArrayTM Plate MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit QuantStudioTM 12K Flex instrument with OpenArrayTM block (AccuFillTM System)
Catalog Number A41237
Publication Number MAN0017952 Revision C.0
For Research Use Only. Not for use in diagnostic procedures.

Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566 USA For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0017952

Revision

Date

C.0

4 January 2021

Description
� Updated instructions to order Custom TaqManTM OpenArrayTM Plates.
� Corrected information for TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate (see Table 3 on page 9). Provided additional information about the number of samples and controls.
� Updated the descriptions of the controls.
� Updated the preamplification reagent to MegaplexTM Preamp Primers, RTM (Cat. No. A41374).
� Specified that the KingFisherTM Flex script is downloaded from the MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit product page.
� Added instructions to review the amplification plot when reviewing results.
� Added approximate Ct ranges for controls. � Corrected the name of the plate in "OpenArrayTM plate assembly and handling
errors" on page 43.

B.0

09 September 2019

� Updated product information for TaqManTM OpenArrayTM Respiratory Tract

Microbiota Plate and controls

� Updated data analysis recommendations

� Updated nucleic acid isolation kit product information and workflow

� Removed Early Access designation

A.0

09 November 2018

New document.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Biomek is a trademark of Beckman Instruments, Inc. Beckman Coulter is a trademark of Beckman Coulter, Inc.
�2021 Thermo Fisher Scientific Inc. All rights reserved.

Contents
CHAPTER 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 TaqManTM assays for respiratory tract microbiota profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
TaqManTM assays included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional TaqManTM assays for respiratory tract microbiota profiling . . . . . . . . . . . . . . . 8 TaqManTM OpenArrayTM Plate products and formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate . . . . . . . . . . . . . . . . . . . . . . . . 9 Custom TaqManTM OpenArrayTM Plate formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Materials required but not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Materials required for nucleic acid isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Materials required for preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Materials required for OpenArrayTM plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Materials required for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Optional controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control . . . . . . . . . . . 14 TaqManTM Universal Extraction Control Organism (B. atrophaeus) . . . . . . . . . . . . . . . . 15 TaqManTM Respiratory Tract Microbiota Amplification Control . . . . . . . . . . . . . . . . . . . . 15 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
CHAPTER 2 Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra
Nucleic Acid Isolation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Before first use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Set up the KingFisherTM Flex instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Reconstitute TaqManTM Universal Extraction Control Organism (B. atrophaeus) . . . . . . . . . 18 Set up the processing plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Set up Sample Plate, then start processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Continue processing to bind, wash, and elute the nucleic acid . . . . . . . . . . . . . . . . . . . . . . . 19

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Contents
CHAPTER 3 Perform preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Perform preamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Dilute the preamplified sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
CHAPTER 4 Prepare and run respiratory tract microbiota profiling
experiments with OpenArrayTM plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Workflow: Microbiota profiling experiments with OpenArrayTM plates . . . . . . . . . . . . . . . . . . 25 One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Set up default folders and software preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Obtain TPF files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software . . . 27 Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate . . . . . . . . . . . . . . . . . . 28 Set up the AccuFillTM instrument and software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument . . . . . . . . . . . . 30 Seal the OpenArrayTM plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument . . . . . . . . . . . . . . . . . 32 Check the quality�control images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
CHAPTER 5 Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Select analysis software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Export data (if necessary) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Considerations for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Approximate Crt range for controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Fields for reviewing results with pivot tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Troubleshooting: Nucleic Acid Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Troubleshoot unexpected Crt values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Troubleshoot with cycling and imaging run images (QC images) . . . . . . . . . . . . . . . . . . . . . . 41 AccuFillTM instrument plate loading errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 OpenArrayTM plate assembly and handling errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Contents
APPENDIX C Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Symbols that may be displayed on the instrument, in the software, or in this guide . . . . . 48 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Overview

Product description
TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate (Cat. No. A41237) is an efficient, easy-to-use fixed-content OpenArrayTM plate for the characterization of key respiratory tract microbial targets. The plate includes TaqManTM assays that have been optimized for detection of respiratory tract viral and bacterial nucleic acid. The plate also includes control assays for TaqManTM Universal Extraction Control Organism (B. atrophaeus), TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control, and the human RNase P RPPH1 gene. For a complete list of assays included in the plate, see "TaqManTM assays included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate" on page 6.
The assays perform well with total nucleic acid that is isolated from nasopharyngeal swab, nasopharyngeal aspirate, and bronchoalveolar lavage (BAL) research samples using the MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit.
TaqManTM assay designs and assay target sequences have undergone rigorous bioinformatics selection and analysis to maximize strain coverage and minimize potential for off-target cross-reactivity. Qualified TaqManTM assays have undergone performance testing to ensure that results are accurate with high levels of sensitivity and specificity.

TaqManTM assays for respiratory tract microbiota profiling

TaqManTM assays included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate

The following TaqManTM assays are included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate.
The assays can also be ordered in a Custom TaqManTM OpenArrayTM Plate.

Table 1 Assays for respiratory tract microbiota

Target organism Bacteria Bordetella bronchiseptica / parapertussis / pertussis Bordetella pertussis Chlamydophila pneumoniae Haemophilus influenzae

Assay name
Bordetella B.pertussis C.pneumoniae H.influenzae

Nucleic acid type

Assay ID

DNA
DNA DNA DNA

Ba06439624_s1
Ba06439623_s1 Ba06439616_s1 Ba06439625_s1

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview TaqManTM assays for respiratory tract microbiota profiling

Table 1 Assays for respiratory tract microbiota (continued)

Target organism

Assay name

Klebsiella pneumoniae

K. pneumoniae

Legionella pneumophila

L.pneumophila

Mycoplasma pneumoniae

M.pneumoniae

Staphylococcus aureus

S.aureus

Streptococcus pneumoniae

S.pneumoniae

Virus

Adenovirus

AdV_1of2

Adenovirus

AdV_2of2

Human Bocavirus

HBoV

Human Coronavirus 229E

CoV_229E

Human Coronavirus HKU1

CoV_HKU1

Human Coronavirus NL63

CoV_NL63

Human Coronavirus OC43

CoV_OC43

Human Enterovirus (pan assay)

EV_pan

Human Enterovirus D68

EV_D68

Human Metapneumovirus (hMPV)

hMPV

Human Parainfluenza virus 1

hPIV1

Human Parainfluenza virus 2

hPIV2

Human Parainfluenza virus 3

hPIV3

Human Parainfluenza virus 4

hPIV4

Human Respiratory Syncytial Virus A (RSVA) RSVA

Human Respiratory Syncytial Virus B (RSVB) RSVB

Human Rhinovirus 1/2

RV_1of2

Human Rhinovirus 2/2

RV_2of2

Human herpesvirus 3 (HHV3 � Varicella zoster Virus)

HHV3

Human herpesvirus 4 (HHV4 � Epstein-Barr HHV4 Virus)

Nucleic acid type DNA DNA DNA DNA DNA

Assay ID Ba04932083_s1 Ba06439617_s1 Ba06439620_s1 Ba04646259_s1 Ba06439619_s1

DNA DNA DNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA DNA
DNA

Vi99990001_po Vi99990002_po Vi99990003_po Vi06439671_s1 Vi06439674_s1 Vi06439673_s1 Vi06439646_s1 Vi06439631_s1 Vi06439669_s1 Vi99990004_po Vi06439642_s1 Vi06439672_s1 Vi06439670_s1 Vi99990005_po Vi99990014_po Vi99990015_po Vi99990016_po Vi99990017_po Vi06439647_s1
Vi06439675_s1

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview TaqManTM assays for respiratory tract microbiota profiling

Table 1 Assays for respiratory tract microbiota (continued)

Target organism

Assay name

Human herpesvirus 5 (HHV5 � Cytomegalovirus)

HHV5

Human herpesvirus 6 (HHV6)

HHV6

Influenza A

Flu_A_pan

Influenza A/H1-2009

Flu_A_H1

Influenza A/H3

Flu_A_H3

Influenza B

Flu_B_pan

Table 2 Assays for respiratory tract microbiota controls

Control name
TaqManTM Universal Extraction Control Organism (B. atrophaeus)
TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control
Human RNase P RPPH1 gene

Assay name B.atrophaeus Xeno RPPH1

Nucleic acid type DNA
DNA RNA RNA RNA RNA

Assay ID Vi06439643_s1
Vi06439627_s1 Vi99990011_po Vi99990009_po Vi99990010_po Vi99990012_po

Nucleic acid type DNA RNA DNA

Assay ID Ba06596576_s1 Ac00010014_a1 Hs04930436_g1

Additional TaqManTM assays for respiratory tract microbiota profiling

The following TaqManTM assays are not included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate. For respiratory tract microbiota profiling using these assays, use a Custom TaqManTM OpenArrayTM Plate.

Target organism Bacteria Bordetella holmesii Coxiella burnetii Moraxella catarrhalis Fungus Pneumocystis jirovecii Virus Human Parechovirus Measles virus Middle East Respiratory Syndrome coronavirus (MERS)

Assay name

Nucleic acid type

Assay ID

B.holmesii C.burnetii M.catarrhalis

DNA DNA DNA

Ba06439621_s1 Ba06439618_s1 Ba06439622_s1

P.jirovecii

DNA

Fn06439626_s1

HPeV Measles MERS_CoV

RNA RNA RNA

Vi99990006_po Vi99990013_po Vi06439644_s1

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview TaqManTM OpenArrayTM Plate products and formats

(continued)
Target organism
Mumps virus
Severe Acute Respiratory Syndrome coronavirus (SARS)

Assay name Mumps SARS_CoV

Nucleic acid type RNA RNA

Assay ID Vi06439657_s1 Vi06439634_s1

TaqManTM OpenArrayTM Plate products and formats

TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate
The TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate (Cat. No. A41237) contains pre-plated, dried down TaqManTM assays for respiratory tract microbiota profiling. For the complete lists of assays included with the plate, see "TaqManTM assays included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate" on page 6.

Contents and storage
Table 3 TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate (Cat. No. A41237)

Component
TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate

Amount 1 plate

Array format 112

Storage �25�C to �15�C

Assays to the 33 microbial targets and the TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control are plated in triplicate. Assays to the TaqManTM Universal Extraction Control Organism (B. atrophaeus) and human RNase P RPPH1 gene control are plated in duplicate. Each TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate can be used to run 23 samples and one control sample.

Custom TaqManTM OpenArrayTM Plate formats

Custom TaqManTM OpenArrayTM Plate contain pre-plated, dried down TaqManTM assays for respiratory tract microbiota profiling.

Array format 56 112 168

Number of assays 56 112 168

Maximum number of samples 48 24 16

Note: We recommend at least three technical replicates of each reaction.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview TaqManTM OpenArrayTM Plate products and formats

Configure and order Custom TaqManTM OpenArrayTM Plates
1. Go to thermofisher.com/order/custom-array.
2. For array type, select TaqManTM OpenArrayTM Real-Time PCR Inventoried Assays Format.
3. (Optional) In the table, click View Layout to preview the layout of the plate.
4. In the table, click Select to configure a plate with the desired array format. The Custom Array Configurator screen displays.

1 Array name 2 Import Your Assay List 3 Save Your Array 4 Complete Your Design
5. Enter the custom array name in the Array name text field.
6. Click Import Your Assay List, then upload or copy-paste the assay information:
� Under Upload a list of Assay IDs, click Choose File, then select a tab-delimited text file (TXT) containing Assay IDs. or
� Under Enter a list of Assay IDs, paste the Assay IDs, then click Import Entered List.
7. Follow the onscreen instructions to configure the assays on the plate.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview Materials required but not supplied

8. (Optional) Click Save Your Array at any time to save the array configuration to your Thermo Fisher Scientific account.
9. When the plate is configured, click Complete Your Design, then follow the onscreen instructions to complete the order.

Materials required but not supplied

Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.

Materials required for nucleic acid isolation
Nucleic acid isolation kit
Table 4 MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit (Cat. No. A42356)

Component Binding Solution Wash Solution Elution Solution Nucleic Acid Binding Beads Proteinase K Enzyme Mix

Amount 60 mL 100 mL 10 mL 2 mL 1 mL 5 mL

Storage 15�25�C �25�C to �15�C

Additional materials
Item
Instrument and equipment KingFisherTM Flex Magnetic Particle Processor 96DW with deep-well heat block Adjustable micropipettors Multi-channel micropipettors Plastics and consumables KingFisherTM Deep-Well 96 Plate KingFisherTM 96 KF microplate (200 �L) KingFisherTM 96 tip comb for DW magnets

Source
5400630 MLS MLS
95040450 97002540 97002534

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview Materials required but not supplied

(continued) Item
Conical Tubes (15 mL) Conical Tubes (50 mL) Nonstick, RNase-Free Microfuge Tubes, 1.5 mL Nonstick, RNase-Free Microfuge Tubes, 2.0 mL MicroAmpTM Clear Adhesive Film Filtered micropipettor tips Reagent reservoirs Reagents Ethanol, 100% (molecular biology grade) Nuclease-free Water Universal Transport Media, for preparation of negative extraction control (Optional) 1X PBS (1X), pH 7.4, for reconstitution of TaqManTM Universal Extraction Control Organism (B. atrophaeus)

Source AM12500 AM12501 AM12450 AM12475 4306311
MLS MLS
MLS AM9932, or equivalent Fisher Scientific 22-031-14, or
equivalent
10010023

Materials required for preamplification

Item Instrument and equipment Thermal cycler, one of the following (or equivalent): � VeritiTM Thermal Cycler, 96-well standard block � SimpliAmpTM Thermal Cycler � ProFlexTM PCR System
Microcentrifuge Vortex mixer Micropipettes Tubes, plates, and other consumables
MicroAmpTM Optical 96-Well Reaction Plate
MicroAmpTM Clear Adhesive Film Aerosol-resistant barrier pipette tips

Source
Contact your local sales office
MLS MLS MLS
N8010560, or equivalent; see thermofisher.com/plastics
4306311 MLS

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 1 Overview Materials required but not supplied

(continued) Item
Disposable gloves Reagents Genomic DNA/RNA MegaplexTM Preamp Primers, RTM TaqPathTM 1-Step RT-qPCR Master Mix, CG Nuclease-free Water
Materials required for OpenArrayTM plate workflow
Item Instruments, software, and equipment OpenArrayTM Sample Tracker Software QuantStudioTM 12K Flex OpenArrayTM Plate Press 2.0 QuantStudioTM 12K Flex Real-Time PCR Instrument with OpenArrayTM block (AccuFillTM System) Centrifuge, capable of spinning sample plates at 1,000 rpm Plates and accessories OpenArrayTM 384-well Sample Plates, black BiomekTM Seal and Sample Foil Lids (for pre-plating step) OpenArrayTM AccuFillTM System Tips QuantStudioTM 12K Flex OpenArrayTM Accessories Kit[2] Forceps Reagents Preamplified genomic DNA/RNA (Optional) TaqManTM Respiratory Tract Microbiota Amplification Control
OpenArrayTM plates with TaqManTM Assays

Source MLS
See page 17 A41374 A15299
AM9937, or equivalent
Source
--[1] A24945 4471090
MLS
4482221 Beckman CoulterTM
538619 4458107 4469576
MLS
See page 21 A39178
� A41237 � Custom ordered[3]

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

1 Chapter 1 Overview Optional controls

(continued)

Item

Source

TaqManTM OpenArrayTM Real-Time PCR Master Mix

4462164

Ethanol

MLS

[1] Included with the QuantStudioTM 12K Flex Software. [2] Each kit contains the items needed to assemble up to 10 plates: 12 lids and plugs, 12 immersion fluid syringes, and 2 carriers. Each
custom OpenArrayTM plate order is shipped with accessories kits. [3] See "CustomTaqManTM OpenArrayTM Plate formats" on page 9.

Materials required for data analysis

Item Software, select one of the following: Relative Quantification Application (recommended)
QuantStudioTM 12K Flex Software

Source
apps.thermofisher.com Included with
QuantStudioTM 12K Flex Real�Time PCR System

Optional controls

Control

Purpose

How to use

TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control

Synthetic RNA control for RNA recovery, reverse transcription, preamplification, and PCR

Nucleic acid isolation: Add to samples along with the Binding/Bead Mix

TaqManTM Universal Extraction Control Organism (B. atrophaeus)

Lyophilized organism control for nucleic acid extraction and purification

Nucleic acid isolation: Standalone sample, or add to samples after Enzyme Mix

TaqManTM Respiratory Tract Microbiota Amplification Control

DNA plasmid control for real- Real-time PCR: Stand-alone

time PCR

sample added directly to the

plate

Cat. No. A39179 A39180 A39178

TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control
TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control is a synthetic RNA control that serves as an exogenous process control for nucleic acid isolation and RNA recovery, reverse transcription, preamplification, and PCR. The control is used with the proprietary TaqManTM assay for XenoTM sequences, which is included in the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate.
TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control is supplied at a concentration of 10,000 copies/�L. During nucleic acid isolation, 10 �L of the control can be added to each test sample along with the nucleic acid binding reagents (Binding Solution). When carried through the

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

1 Chapter 1 Overview Optional controls
respiratory tract microbiota workflow, the control is used to monitor nucleic acid recovery, RNA reverse transcription, cDNA preamplification, and PCR. The control can be used to identify sample-specific amplification inhibition, which reduces the likelihood of false negatives and provides confidence that results are accurate. It is recommended that the control is added to each sample during nucleic acid isolation.
TaqManTM Universal Extraction Control Organism (B. atrophaeus)
TaqManTM Universal Extraction Control Organism (B. atrophaeus), serves as a process control for cell lysis and nucleic acid recovery. The control is used with the proprietary TaqManTM assay for Bacillus atrophaeus sequences.
Like other gram-positive bacteria, Bacillus atrophaeus has thick cell walls than can be difficult to lyse. This characteristic makes gram-positive bacteria an ideal control to monitor the efficiency of cell lysis and subsequent nucleic acid recovery.
TaqManTM Universal Extraction Control Organism (B. atrophaeus) is supplied lyophilized with a quantity of 1 � 109 copies/vial, and is reconstituted in 200 �L of 1X PBS (1X), pH 7.4 to a final concentration 5 � 106 copies/�L. During nucleic acid isolation, 10 �L of the control is processed as a stand-alone sample in a background of universal transport media. It can be added to the negative extraction control, and may also be added to one or more test samples at the start of the extraction process. The control is carried through the remainder of the workflow with the test samples. It is recommended that at least one stand-alone control sample is run per extraction plate.
TaqManTM Respiratory Tract Microbiota Amplification Control
TaqManTM Respiratory Tract Microbiota Amplification Control contains a linearized multi-target plasmid with target sequences for each available respiratory tract microbiota profiling assay. The plasmid also contains target sequences for the TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control, the TaqManTM Universal Extraction Control Organism (B. atrophaeus), and the human RNase P RPPH1 gene. It can be included in respiratory tract microbiota profiling experiments as a positive control for panel-specific amplification.
TaqManTM Respiratory Tract Microbiota Amplification Control is supplied at a concentration of 1 � 105 copies/�L. During real-time PCR, 2.5 �L of the control is used as a standalone sample in 2 wells of the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate. The control can be used if needed to verify assay performance and to help with troubleshooting.
Note: The amplification control RV target sequence is a perfect match to the RV_1of2 assay target, and contains a mismatch with the RV_2of2 assay target. Lower Amp Scores and Cq confidence scores can be noted for RV_2of2 versus RV_1of2.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

1 Chapter 1 Overview Workflow
Workflow
Start with bronchoalveolar lavage, nasopharyngeal swab, or nasopharyngeal aspirate samples
Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit (page 17)
Perform preamplification (page 21)
Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates (page 24)
Analyze data (page 35)

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra Nucleic Acid

Isolation Kit

For required materials, see "Materials required for nucleic acid isolation" on page 11.
Procedural guidelines
� Perform all steps at room temperature (20�25�C) unless otherwise noted. � Ensure that Nucleic Acid Binding Beads remain in a homogeneous suspension while pipetting.
Vortex beads before use.
Before first use of the kit
� Download the KingFisherTM Flex script MVP_Ultra_Flex from the MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit (Cat. No. A42356) product page, then install it on the instrument. See the instrument user guide for instructions to install the script.
� Prepare fresh 80% Ethanol using 100% absolute Ethanol and Nuclease-free Water, sufficient for 1.5 mL per sample, plus 10% overage.
Set up the KingFisherTM Flex instrument
� Ensure that the KingFisherTM Flex instrument has the appropriate magnetic head and heat block installed. � 96 deep-well magnetic head � 96 deep-well heat block
� Ensure that the MVP_Ultra_Flex script is installed on the instrument.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 2 Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit Reconstitute TaqManTM Universal Extraction Control Organism (B. atrophaeus)

Reconstitute TaqManTM Universal Extraction Control Organism (B. atrophaeus)
Use of the TaqManTM Universal Extraction Control Organism (B. atrophaeus) is optional. 1. Remove metal fastener from vial using tweezers and place vial on ice. 2. Remove rubber stopper from vial, then add 200 �L 1X PBS (1X), pH 7.4 to the vial. 3. Replace the rubber stopper, then vortex the tube to mix. 4. Transfer reconstituted sample to a 1.5-ml tube, then store on ice or at 4�C. Note: Store the reconstituted control at 4�C for up to 48 hours. For long term storage, store the reconstituted control at �80�C to �20�C for up to 4 months. Mix well to resuspend before use.
The final concentration of the control is 5 � 106 copies/�L.

Set up the processing plates

Set up the processing plates outside the instrument according to the following table. Cover the plates with a temporary seal, then store at room temperature for up to 1 hour while you set up Sample Plate.

Plate type

Plate position

Plate ID

Wash 1 Plate

3 Deep well[1]
4

Wash 2 Plate Wash 3 Plate

Elution Plate

Standard[3]

Tip Comb

[1] KingFisherTM Deep-Well 96 Plate [2] The elution volume can be increased to a maximum of 100 �L. [3] KingFisherTM 96 KF microplate

Reagent
Wash Solution 80% Ethanol 80% Ethanol Elution Solution 96DW Tip Comb

Volume per well
1000 �L 1000 �L 500 �L 60 �L[2]
--

Set up Sample Plate, then start processing
(Optional) Reconstitute TaqManTM Universal Extraction Control Organism (B. atrophaeus) before use in step 3 (see page 18).
1. Swirl the bottle of Enzyme Mix, then place on ice.
2. Add 50 �L of Enzyme Mix to each well in a KingFisherTM Deep-Well 96 Plate (Sample Plate).

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 2 Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit Continue processing to bind, wash, and elute the nucleic acid

3. Add samples and controls to the appropriate well containing Enzyme Mix.

Sample or control Sample Negative Extraction Control (NEC)
(Optional) TaqManTM Universal Extraction Control Organism (B. atrophaeus)

Instructions Add 200�400 �L of sample to a well.
Add 200�400 �L of Universal Transport Media to a well.
� Combine 10 �L of reconstituted control with 390 �L of Universal Transport Media in a well. or
� Add 10 �L of reconstituted control to one or more sample wells.

4. On the KingFisherTM Flex instrument, select the MVP_Ultra_Flex script, then press Start. 5. Follow the instrument prompts to load sample and processing plates, then press Start. Proceed immediately to the next step.

Continue processing to bind, wash, and elute the nucleic acid
1. During the enzyme treatment incubation on the instrument, prepare the Binding/Bead Mix. a. Vortex the tube of Nucleic Acid Binding Beads to fully resuspend the beads.
b. Combine the following components for the required number of samples, plus 10% overage. IMPORTANT! Binding Solution is viscous. Pipet slowly to avoid bubbles and to ensure that the correct volume is delivered.

Component Binding Solution Nucleic Acid Binding Beads (Optional) TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control
Total

Volume per sample 530 �L 20 �L
10 �L
550 �L or 560 �L

2. Gently invert the Binding/Bead Mix 5 times to mix, then store at room temperature until the next step.
3. When prompted by the instrument (approximately 20 minutes after the start of the script), remove the Sample Plate from the instrument.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 2 Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit Continue processing to bind, wash, and elute the nucleic acid

4. Add 10 �L of Proteinase K to each sample in the Sample Plate.
Note: Add the Proteinase K to the sample separately from and before the Binding/Bead Mix. Combining the reagents, or adding in a different order can affect nucleic acid recovery.
5. Gently invert the Binding/Bead Mix 5 times to mix, then use a manual pipet (single or multichannel) to dispense the appropriate volume to each sample and control well in the Sample Plate. � 550 �L: Binding/Bead Mix only or � 560 �L: Binding/Bead Mix + TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control
IMPORTANT! Binding/Bead Mix is viscous. Pipet slowly to avoid bubbles and to ensure that the correct volume is delivered. Invert the Binding/Bead Mix regularly to avoid bead settling.
6. Return Sample Plate to the instrument, then press Start to resume the script.
7. When processing is complete (~30 minutes after adding Binding/Bead Mix), remove Elution Plate from instrument. The purified nucleic acid is in Elution Plate.
8. Transfer the nucleic acid samples to a 96well storage plate or seal Elution Plate.
Store nucleic acid samples on ice for immediate use or at �20�C for longer-term storage.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Perform preamplification

For required materials, see "Materials required for preamplification" on page 12.

Good laboratory practices for PCR and RT-PCR
� Wear clean gloves and a clean lab coat. � Do not wear the same gloves and lab coat that you have previously used when handling amplified products or preparing samples.
� Change gloves if you suspect that they are contaminated. � Maintain separate areas and dedicated equipment and supplies for:
� Sample preparation and reaction setup. � Amplification and analysis of products. � Do not bring amplified products into the reaction setup area. � Open and close all sample tubes carefully. Avoid splashing or spraying samples. � Keep reactions and components capped as much as possible. � Use a positive-displacement pipettor or aerosolresistant barrier pipette tips. � Clean lab benches and equipment periodically with 10% bleach solution or DNA decontamination solution.

Perform preamplification

Note: Preamplification of the TaqManTM Respiratory Tract Microbiota Amplification Control is not recommended.

1. Prepare PreAmp Reaction Mix: Combine the following components for the number of required reactions plus 10% overage, then mix thoroughly by pipetting up and down.

Component TaqPathTM 1-Step RT-qPCR Master Mix, CG MegaplexTM Preamp Primers, RTM[1]

Volume per reaction 2.5 �L 2.5 �L

[1] MegaplexTM Preamp Primers, RTM contains primers for all of the respiratory tract microbiota assays, plus the TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control and the RNase P RPPH1 gene. It does not contain primers for the TaqManTM Universal Extraction Control Organism (B. atrophaeus).

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 3 Perform preamplification Dilute the preamplified sample

2. Distribute PreAmp Reaction Mix, then Nuclease-free Water or sample nucleic acid to the appropriate wells of a 96-well plate.

Component

Sample reaction

No-template control (NTC) reaction

PreAmp Reaction Mix

5 �L

Sample DNA or NEC

5 �L

Nuclease-free Water

5 �L

Total volume per reaction

10 �L

3. Seal the plate with adhesive film.

4. Gently vortex the plate for 10 seconds to mix, then briefly centrifuge to bring contents to the bottom of the wells.

5. Place the plate in a thermal cycler that is programmed with the following thermal cycling conditions, then start the run.

Stage Hold Hold Hold
Cycling (14 cycles)
Hold Hold

Step UNG incubation[1] Reverse transcription
Activation Denaturation Annealing/ Extension Inactivation
--

Temperature 25�C 50�C 95�C 95�C 60�C 99.9�C 4�C

Time 2 minutes 30 minutes 2 minutes 15 seconds 2 minutes 10 minutes
Hold

[1] Heat-labile UNG is completely inactivated during the initial ramp to 95�C.

6. Store the plate on ice until dilution for PCR (see "Dilute the preamplified sample" on page 22).

Dilute the preamplified sample
To determine dilution volumes, first determine the total volume of diluted preamplified sample that is required for PCR (see Chapter 4, "Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates"). We recommend that you prepare only the volume of diluted preamplified sample that is required for your experiment. The undiluted preamplified sample can be stored at �20�C long term.
1. Vortex, then briefly centrifuge the plate that contains the completed preamplification sample reactions.
2. Remove the adhesive film from the plate.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 3 Perform preamplification Dilute the preamplified sample

3. Prepare a 1:10 dilution of the preamplified samples in a new 96-well plate. a. Transfer the desired volume of the preamplified samples to a new 96-well plate (for example, 2 �L).
b. Add the appropriate volume of Nuclease-free Water to each sample and control well (for example, 18 �L).
4. Seal the plate with new adhesive film.
5. Vortex the plate for 10 seconds, then briefly centrifuge.
6. Proceed directly to PCR (see Chapter 4, "Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates" ).
Seal the plate that contains the unused portion of the undiluted preamplified samples, then store at �20�C.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Prepare and run respiratory tract microbiota profiling experiments

with OpenArrayTM plates

For required materials, see "Materials required for OpenArrayTM plate workflow" on page 13. This chapter contains brief procedures. For detailed procedures, see the following documentation.

Document
QuantStudioTM 12K Flex Real�Time PCR System: OpenArrayTM Experiments User Guide
OpenArrayTM Sample Tracker Software Quick Reference
OpenArrayTM AccuFillTM System User Guide

Pub. No. 4470935 4460657 4456986

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Workflow: Microbiota profiling experiments with OpenArrayTM plates

Workflow: Microbiota profiling experiments with OpenArrayTM plates

Preamplified genomic DNA/RNA (Chapter 2, "Isolate nucleic acid using MagMAXTM Viral/Pathogen Ultra Nucleic Acid Isolation Kit")

Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software (page 27)

Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate (page 28)

Set up the AccuFillTM instrument and software (page 29)

Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument (page 30)

Seal the OpenArrayTM plate (page 31)

Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument (page 32)

Check the quality�control images (page 33)

One-time procedures
Set up default folders and software preferences
This procedure simplifies the file locations used in the AccuFillTM software. Set up the default file locations and preferences before using the OpenArrayTM AccuFillTM system for the first time. You must be logged in as an administrator.
1. Create the following four folders in a convenient location on the same computer drive as the AccuFillTM software: � TPF Files � Sample Tracker 96-well Input � Sample Tracker 384-well CSV Files � Loaded TPF Files

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates One-time procedures

2. (Optional) Copy a template file into the OpenArrayTM Sample Tracker Software folder.
� Navigate to this folder on your computer: <drive>:\Program Files (x86)\Applied Biosystems\OpenArray Sample Tracker\examples.
� Copy the 96Well Sample Plate 1.csv template file, which is provided with the OpenArrayTM Sample Tracker Software.
� Paste the template file into the Sample Tracker 96well Input folder.

3. In the OpenArrayTM Sample Tracker Software, select View4Preferences, then enter the following preferences:

Field Experiment Type OpenArrayTM Plate
Pipettor Import Data Directory Export Data Directory

Selection Gene Expression Select the OpenArrayTM format that will be run most often, such as Gene Expression � 112. Fixed or Adjustable tip spacing Sample Tracker 96-well Input Sample Tracker 384-well CSV Files

4. In the AccuFillTM software, select Instrument4Edit Preferences, then: a. Select Require Sample Integration.

b. Select the folders indicated in this table:

AccuFillTM folder
OpenArrayTM Plate File Input Folder

Default folder TPF Files

Sample Plate File Folder
Loaded OpenArrayTM Plate File Folder

Sample Tracker 384-well CSV Files
Loaded TPF Files

Folder contents
TPF files for the OpenArrayTM plates, with assay name and location
CSV 384-well sample plate layout files
Integrated TPF files generated during processing with the AccuFillTM software.

5. In the QuantStudioTM 12K Flex Software, select Tools4Preferences4OpenArray, then select the Loaded TPF Files folder for the software Setup Folder.
Note: If the QuantStudioTM 12K Flex Software is not on the same computer as the AccuFillTM software, transfer the loaded TPF files to the computer running the QuantStudioTM 12K Flex Software.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software

Obtain TPF files
Go to thermofisher.com/OA-platefiles to obtain the TPF files for the TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate.
Set up the optimized folder locations and software preferences before downloading TPF files to your computer. See "Set up default folders and software preferences" on page 25.

Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software

Before generating 384well sample plate layouts, see "One-time procedures" on page 25 to complete the following tasks:
� Set up optimized folder locations and software preferences. � Download the TPF files for the OpenArrayTM plates into the TPF Files folder.
1. Using a spreadsheet program, create a 96well sample CSV file. a. Navigate to the following folder, then open the 96-Well Sample Plate 1.csv template that is provided with the OpenArrayTM Sample Tracker Software. <drive>:\Program Files (x86)\Applied Biosystems\OpenArray Sample Tracker\examples

b. Save As the template as a new 96well sample CSV file. Save your 96well sample CSV file in the Sample Tracker 96-well Input folder.

c. Enter or copy the sample names into your 96well sample CSV file.

2. Open the OpenArrayTM Sample Tracker Software.

3. In the Properties screen, select Gene Expression for Experiment Type, then select the appropriate settings for OpenArrayTM Plate and Pipettor.

4. In the Samples screen, click you created in step 1.

Import, then select and import your 96well sample CSV file that

5. In the Sample Mapping screen, confirm that the samples for a single OpenArrayTM plate are assigned to one color.

Note: If necessary, correct the OpenArrayTM Plate and Pipettor settings in the Properties screen.

6. In the Sample Mapping screen, click the 384-Well Plate tab, then click Export4Export *.csv.
7. Select 384-Well Plate (for AccuFill), enter a file name, then save the exported file.
Plate layouts for the 384-well sample plates are saved to individual CSV files in the Sample Tracker 384-well CSV Files folder.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate

Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate

IMPORTANT! The 4 � 12 area(s) of the OpenArrayTM 384-well Sample Plate being filled must match the area(s) designated in the OpenArrayTM Sample Tracker Software for that set of samples.

1. Remove an OpenArrayTM plate from the freezer and set it aside. Allow it to come to room temperature in its unopened sleeve (~15 minutes).
The OpenArrayTM plate must be completely thawed before transferring reactions to it from the OpenArrayTM 384-well Sample Plate created in this section.

2. Gently swirl the contents of the TaqManTM OpenArrayTM Real-Time PCR Master Mix to thoroughly mix. Do not invert the bottle.

3. Following the plate layout designated in the OpenArrayTM Sample Tracker Software, add master mix, then DNA samples, to the wells of an OpenArrayTM 384-well Sample Plate.
(Optional) Use the TaqManTM Respiratory Tract Microbiota Amplification Control in place of diluted preamplified sample, as a positive amplification control sample.

Component TaqManTM OpenArrayTM Real-Time PCR Master Mix Diluted preamplified sample Total reaction volume

Volume per well 2.5 �L 2.5 �L 5.0 �L

Volume per sample[1] 5.5 �L 5.5 �L 11.0 �L

[1] Volume includes 10% overage.

4. Thoroughly mix each PCR reaction by pipetting up and down or by using the "mix" function on a multi-channel pipette.

5. Seal the OpenArrayTM 384-well Sample Plate with an aluminum foil seal, remove the foil flap, then mark the edges of the filled 4 � 12 area with a pen.

6. Centrifuge the plate at 1,200 � g for 1 minute.

7. Score the foil along the lines that were marked before centrifuging. Do not remove the foil from the scored area at this time.

If you make a sample layout error before the AccuFillTM procedure � Repeat "Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software" on page 27 with a corrected 96well sample CSV file.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Set up the AccuFillTM instrument and software

Set up the AccuFillTM instrument and software
IMPORTANT! Do not use OpenArrayTM AccuFillTM System Tips that exceed the expiration date (shown on the outer box that contains the tip trays).
1. In the OpenArrayTM AccuFillTM software, click Setup and Load. The Setup Load Information window appears.

1 Use Sample Integration checkbox; select to integrate TPF files and the 384-well sample plate CSV file. 2 Browse button; click to locate and select the 384-well sample plate CSV file. The button is displayed only if Use
Sample Integration is selected. 3 Browse buttons; click to locate and select the TPF files for the OpenArrayTM plates that will be placed in the
corresponding Plate Holder Position on the deck of the AccuFillTM instrument. The buttons are displayed only if Use Sample Integration is selected. 4 Plate Holder Position corresponding to the position of the OpenArrayTM plate on the deck of the AccuFillTM instrument.
2. Configure the Loading Information pane for sample integration using the 384-well sample plate CSV file and TPF files. a. In the Loading Information pane (top section of the window), ensure that the Use Sample Integration checkbox is selected.
b. Click Browse to the right of the Sample Plate field, then select the 384-well sample plate CSV file that you generated with the OpenArrayTM Sample Tracker Software in the Sample Tracker 384-well CSV Files folder.
c. Click Browse to the right of the Plate Holder Position of the OpenArrayTM plate, then select the TPF file for the OpenArrayTM plate in the TPF Files folder.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument

3. In the Select Samples to Load pane (bottom section of the window), click the corresponding 4 � 12 area of the 384-well sample plate image, then click Next. The Setup Deck window is displayed.
4. In the AccuFillTM instrument, ensure that: � Tip boxes and tips are loaded as shown in the Setup Deck window. � The lids are removed from the tip boxes. � The waste bin in the instrument is emptied.
5. In the Setup Deck window, confirm that the deck is ready: � Select The tips are configured as shown above. � Select The Waste Bin is empty.

Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument
IMPORTANT! Ensure that the OpenArrayTM plate is thawed and that the entire plate is at room temperature.
1. Prepare the items needed to seal the loaded OpenArrayTM plate (next section).
Note: The OpenArrayTM plate must be sealed promptly after being loaded with the reactions, as described here.
� Ensure that the QuantStudioTM 12K Flex OpenArrayTM Plate Press 2.0 is ready. � Gather and remove from their packaging the following: an OpenArrayTM Lid, plug, syringe with
OpenArrayTM Immersion Fluid, and syringe tip. � Attach the syringe tip to the syringe, carefully push some of the fluid through the tip to remove
air bubbles, then lay the syringe aside.
2. Load the OpenArrayTM plate and the OpenArrayTM 384-well Sample Plate into the AccuFillTM instrument. � OpenArrayTM plate--Remove the plate from its sleeve, then place the plate in the appropriate plate holder position in the instrument. Ensure that the barcode on the OpenArrayTM plate is facing left and the serial number is facing right. � OpenArrayTM 384-well Sample Plate--Place the 384-well sample plate onto the deck of the instrument, then use forceps to peel the foil from the filled area of the plate.
3. Close the door of the AccuFillTM instrument.
4. In the AccuFillTM software Setup Deck window, select the following confirmations: � The OpenArray Plate is in the Plate Holder � Remove foil from the highlighted section of the Sample Plate

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Seal the OpenArrayTM plate

5. Click Load.
6. As soon as the Remove OpenArray Plate window appears, open the instrument door and remove the loaded OpenArrayTM plate.
7. Proceed immediately to seal the OpenArrayTM plate (next section).
Note: For best results, seal the OpenArrayTM plate within 90 seconds of completion of loading to prevent evaporation.

Seal the OpenArrayTM plate

IMPORTANT! Throughout this procedure, handle the OpenArrayTM plate and the OpenArrayTM Case only by the edges.

Note: The OpenArrayTM Case consists of the sealed OpenArrayTM plate and the OpenArrayTM Lid.
1. Place the newly loaded OpenArrayTM plate in the QuantStudioTM 12K Flex OpenArrayTM Plate Press 2.0. Ensure that the barcode is facing left and the serial number is facing right.
2. From the OpenArrayTM Lid, remove the clear protective film from the inside of the lid and the red adhesive-protective strip from around the edge of the lid.

OpenArrayTM Lid 1 Protective film on inside of the lid (remove before sealing) 2 Red adhesive-protective strip (remove before sealing) 3 Protective film on the outside of the lid (remove before running) 4 Notched end (align with serial number on plate)
3. Seat the lid on the OpenArrayTM plate with the lid adhesive against the plate.
4. Engage the press mechanism until the green flashing light changes to a steady green light (approximately 20 seconds).
5. Disengage the press and remove the OpenArrayTM Case.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument

6. While holding the case by its edges, insert the prepared syringe tip into the port in the case, then carefully inject OpenArrayTM Immersion Fluid until the case is filled.
Note:
� Minimize creation of air bubbles when you dispense the fluid. � Leave a small bubble at the fill point to prevent fluid leaks during the instrument run.

The syringe tip must be in front of the array when filling the case with immersion fluid.
7. While holding the case vertically, remove the syringe tip, insert the screw end of the OpenArrayTM plug into the port of the case, then rotate clockwise until the black handle breaks off.
IMPORTANT! To avoid leaking of immersion fluid, hold the case vertically and rotate the plug slowly.
If the plug handle breaks off prematurely, use a Phillips #0 screwdriver to complete this step.
8. If needed, clean the case with a laboratory wipe that has been thoroughly sprayed with ethanol, then dry the case with a clean laboratory wipe.

Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument

You can run up to four OpenArrayTM plates at one time on the QuantStudioTM 12K Flex Instrument.

1. On the QuantStudioTM 12K Flex Instrument touchscreen, touch arm.

to extend the instrument tray

2. Remove the clear protective film from the outside of the OpenArrayTM case (sealed plate + lid).

3. Place the OpenArrayTM case on the tray arm plate adapter.
� Support the case from underneath the tray arm to prevent the case from slipping through the adapter.
� Ensure that the plate barcode and serial number are facing the front of the instrument.

4. Touch to retract the instrument tray arm.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Check the quality�control images

5. In the Home screen of the QuantStudioTM 12K Flex Software, in the Run pane, click OpenArray.
6. In the Select Instrument pane, select your instrument.
7. Click Get Plate IDs to import the barcode of the OpenArrayTM plate. Once the OpenArrayTM serial number appears, the loaded TPF file corresponding to the plate should appear in the Setup File field. If the TPF file does not appear, click Browse, then select the correct loaded TPF file from the Loaded TPF folder.
8. (Optional) Click Browse to change the Experiment File Location.
9. (Optional) Change the software-determined Experiment File Name.
10. Click Start Run.
Note: The instrument pauses at 41 or 42 seconds prior to the end of the run. Wait for the system to complete the run before opening the EDS file.
11. Transfer the EDS file from the instrument to an accessible location for analysis.
12. Check the QC images for loading issues or leaks.

Check the quality�control images

Check the quality�control (QC) images before analysis. Images can be viewed using ImageJ, an open�source software available from the NIH at imagej.nih.gov/ig. For additional information, see Appendix A, "Troubleshooting".

1. In the QuantStudioTM 12K Flex Software named folder for the QC images export.

Export screen, click Browse, then create a uniquely�

IMPORTANT! Create a new folder for images each time. Exporting a second run to the same folder overwrites the images.

2. Click Export QC Images at the bottom of the screen.
3. View the following ROXTM image to check for loading quality issues: � POST-READ_CHANNEL_4.tiff
4. Check the following spotfinding images for leaks or other displaced sample issues. � s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff � s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff
Note: The "cp#" in the image file name refers to array positions 1 through 4 within the instrument.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 4 Prepare and run respiratory tract microbiota profiling experiments with OpenArrayTM plates Check the quality�control images

5. If a problem is found, view the following pre-run spotfinding image to determine whether the issue existed before cycling: � s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff
6. View the following FAMTM images to check for fluorescent abnormalities and to confirm any problem seen in the spotfinding images: � STAGE2_CYCLE1_CHANNEL_1.tiff � STAGE2_CYCLE40_CHANNEL_1.tiff
7. Note any abnormalities found, as well as all other potentially relevant information related to the setup of the run.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Analyze data

Select analysis software

Software
Relative Quantification Application (recommended)[1] Access the application at thermofisher.com/connect.

Analysis option

Crt

QC metrics

QuantStudioTM 12K Flex Software

[1] To perform data analysis using the application, you must export your data. For detailed instructions about exporting data, see "Export data (if necessary)" on page 35.

Export data (if necessary)
Export your data to review it using the pivot table feature of a spreadsheet program. 1. Open an EDS file in the QuantStudioTM 12K Flex Software.
2. In the Experiment Menu pane, click Export.
3. Click Load Export Set (bottom of the screen), select GE_export_setting, then click OK.
4. Select .xlsx from the File Type dropdown list (top-right of the screen).
5. (Optional) Perform any of the following actions to customize the file export. � Click Browse to select a new Export File Location. � Enter a new file name in the Export File Name text field. � Click the Results tab, then select the content to export.
6. Click Start Export (bottom of the screen). If Open file(s) when export is complete is selected, then the file automatically opens. If the option is not selected, navigate to and open the exported XLSX file.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

5 Chapter 5 Analyze data Review results

Review results

Note: These guidelines apply to results from experiments that included three or more technical replicates.

1. In the analysis settings of the software, choose the relative Crt method. The relative Crt method is recommended for dried-down assays. Dried-down assays can reconstitute at different rates, causing a dip in the early cycles of the baseline. Crt can correct for a variable baseline.
� In Relative Quantification Application : a. Click (Analysis Settings). b. In the Cq Settings tab, select Use Crt. c. Click Finish.
� In QuantStudioTM 12K Flex Software-- select Analysis Settings4Ct Settings4Algorithm Settings4Relative Threshold.

2. Review amplification curves (in log or linear view), Crt values, and amplification curve QC metrics (Amp Score and Cq Confidence) for each reaction.

QC metric

Description

Amp Score

A value to indicate the quality of the amplification curve.

Cq Confidence

A value to reflect the reliability of the derived Cq.

3. (Optional) Filter data in the order indicated in the following table.

Note: We encourage testing and establishing your own Crt cut-off value and amplification curve QC metrics for each assay to achieve high sensitivity and specificity.

Parameter to examine
Crt Amp Score
Cq Confidence

Consider filtering out sample data using the following cut-off values
Crt > 28 Amp Score < 1.2 The following assays have a cut-off value of < 1.1
� RNase P (RPPH1) � Human Enterovirus (EV_pan)
Cq Conf < 0.7 Note: The following assays have a cut-off value of < 0.5
� RNase P (RPPH1) � Bordetella holmesii (B. holmesii)

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 5 Analyze data Considerations for data analysis

Note:
� We encourage testing and establishing your own Crt cut-off value for each assay to achieve high
sensitivity and specificity.
� Through-holes with unexpected Crt values can also be identified by reviewing the Amplification
Plot (see "Troubleshoot unexpected Crt values" on page 40).
4. Note replicates with mean Crt 25 and a standard deviation > 0.5. The data from these throughholes might require further review.
5. Ensure that at least half of the replicates amplified adequately and pass your review specifications.
6. Use your preferred method to analyze the data.

Considerations for data analysis

Organisms that are covered by more than one assay
For full strain coverage of adenoviruses or rhinoviruses, two assays are used. A positive result with either or both of the assays indicates a positive result for the organism.

Species-specific assays that are also covered by pan or broad coverage assays

Species-specific assay Considerations for data analysis

Flu A assays

� The Flu_A_pan assay detects Influenza A H1 and H3, for which there are also strain-specific assays.
� Samples that are positive for the Flu_A_H1 or Flu_A_H3 assay typically are positive for the Flu_A_pan assay.

Bordetella assays

� The Bordetella assay detects B. pertussis, B. bronchiseptica, and B. parapertussis strains. Strain-specific assays for B. pertussis and B. holmesii are also available.
� Most samples that are positive for the B. pertussis assay are also positive with the Bordetella assay.

RSV assays
Enterovirus (EV) and rhinovirus (RV) assays

� Samples that are positive for the RSVA assay may be detected at a lower efficiency (a difference of several Crt values) by the RSVB assay.
� The RV assays detect both RV and EV strains whereas the EV assays are specific for EV strains. Thus enterovirus positive samples are detected by both EV and RV assays whereas rhinovirus positive samples are detected only by the RV assays.
� The EV_pan assay detects all human enterovirus species except D68, for which there is a strain-specific EV_D68 assay.
� Samples that are positive for the EV_D68 assay may be detected at a lower efficiency (a difference of several Crt values) by the EV_pan assay.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 5 Analyze data Approximate Crt range for controls

(continued)

Species-specific assay Considerations for data analysis

Bacterial and HHV assays

� It is not unusual to detect M. catarrhalis, H. influenzae, K. pneumoniae, S. pneumoniae, and S. aureus in respiratory samples as these are commensal or transiently commensal upper respiratory tract microbes.
� Due to the high prevalence of human infection with HHV4 (EBV) and HHV6 viruses, these viruses can be detected at low levels in some respiratory samples.

Approximate Crt range for controls

If used as recommended in this guide, the approximate Crt range for control assays are as follows.

Control
TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control
TaqManTM Universal Extraction Control Organism (B. atrophaeus)
TaqManTM Respiratory Tract Microbiota Amplification Control

Approximate Crt range 18�20
21�23 18�21

Fields for reviewing results with pivot tables

To review results using the pivot table feature of a spreadsheet program, you can use the following settings.
Note: For the "Average of" and "StdDev of" summarizations, use the appropriate source field (Crt, Amp Score, or Cq Conf), then choose the calculation type.

Area of pivot table
Filters Columns Rows Values

Fields to add

Target-oriented view

Sample-oriented view

Sample Name[1]

Sample Name

Target Name

Average of Crt StdDev of Crt[2]
Count of Crt

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Chapter 5 Analyze data Fields for reviewing results with pivot tables

(continued)

Area of pivot table

Fields to add

Target-oriented view

Sample-oriented view

Values

Average of Amp Score

Average of Cq Conf

[1] To see individual sample results, select the sample from the dropdown list next to the Sample Name header. [2] A Values field will automatically appear in the Column Labels area.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Troubleshooting

Troubleshooting: Nucleic Acid Isolation

Observation Beads remain in sample after elution
Reduced extraction efficiency of TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control

Possible cause
Excessive amount of recovered host genomic DNA/RNA is preventing nucleic acid separation from the beads.
Proteinase K enzyme was either omitted from the sample or added incorrectly.
TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control added at the wrong step.

Recommended action
Increase elution volume to 100 �L.
Reduce the input volume of starting sample to 200 �L.
Always add Proteinase K enzyme to the sample separately and before adding the Binding/Bead Master mix.
Ensure that the TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control is added to the Binding/Bead Master mix before dispensing into sample wells.

Troubleshoot unexpected Crt values

Observation

Possible cause

Unexpected Crt values in the amplification plot

Unexpectedly low Crt values (<10) -- Signal variation or

noise in early PCR cycles

1 Expected Crt value (noted in most replicates) 2 Unexpected Crt value (too low)

Unexpectedly high Crt values (25�28) -- Sporadic amplification

Recommended action
Review amplification curves, Amp Score, and Cq Confidence values.
Consider filtering Crt values from analysis.
Compare replicates, if available.
Dilute the samples, then repeat the experiment.
Review amplification curves, Amp Score, and Cq Confidence values.
Compare replicates, if available.
Repeat the experiment.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Appendix A Troubleshooting Troubleshoot with cycling and imaging run images (QC images)

Troubleshoot with cycling and imaging run images (QC images)

Many problems with OpenArrayTM results can be diagnosed by examining the quality control (QC) images taken at various points during a cycling/imaging run.
The QC images are fluorescent or reflected light images taken before, during, and after cycling. They may require adjustment to make image features visible. To view the images, we recommend that you install the free software program ImageJ, which allows you to easily manipulate the images in ways that other image viewers cannot.
1. In the QuantStudioTM 12K Flex Software Export screen : a. Click Browse to select a uniquely-named folder for the QC images export.
b. Click Export QC Images (bottom of screen).

IMPORTANT! Select a new folder for images each time; exporting a second run to the same folder overwrites the images.

2. Use ImageJ to view the images of interest.

To...

View image...

Image description

Confirm the identity of BARCODE IMAGE.tiff images within a folder

Reflected light image of the entire OpenArrayTM plate

Evaluate the loading quality

PRE-READ_CHANNEL_4.tiff POST-READ_CHANNEL_4.tiff

Pre- and post-ROXTM dye images

Check for existing contamination on the case and/or heated cover

s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff[1] Pre-run reflected light spotfinding image (used by the software to determine the location of the holes)

Identify potential leaks s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff[1] Mid-run reflected light spotfinding

or other contamination

image

s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff[1] Post-run reflected light spotfinding image

Look at patterns in the fluorescent data (for example, gradients)

STAGEx_CYCLEy_CHANNEL_1.tiff

FAMTM images at a particular cycle (y) of a particular stage (x) of the run.

[1] The "cp#" in the image file name refers to the array position (1�4) within the QuantStudioTM 12K Flex Real-Time PCR Instrument.

3. (Optional) Adjust the images for brightness and/or contrast to make image features visible. a. Open the image in ImageJ.

b. Select Image4Adjust Brightness/Contrast (or press Ctrl+Shift+C).

c. Click Auto or adjust the sliders until the features of interest in the image are visible.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Appendix A Troubleshooting AccuFillTM instrument plate loading errors

AccuFillTM instrument plate loading errors

Observation There are empty through-holes
Turn-holes are repeatedly missed

Possible cause

Recommended action

Insufficient sample was added to the 384-well Sample Plate.

Use proper pipetting techniques. Ensure that there are no air bubbles in the pipette tips after sample aspiration.

Reaction mix (sample + master mix) is not at the bottom of the 384-well Sample Plate.
AccuFillTM instrument is aligned too far to the left or to the right.

Centrifuge the plate at 1,200 � g for 60 seconds.
Contact your local field service engineer.

Systematic loading problems can occur with the AccuFillTM instrument, which indicates a need for service. For example, when turn-holes are repeatedly missed across multiple subarrays, service is required. Turnholes are where the AccuFillTM instrument changes direction during sample loading.

Entire subarrays are missing

Turn holes Start points Stop points

The sample/master mix was not added to particular wells in the 384-well Sample Plate.
Stuck tip mandrel on AccuFillTM instrument may need cleaning.

Visually inspect the plate to ensure that the wells have sample/master mix.
Contact your local field service engineer.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Observation Entire subarrays are missing

Appendix A Troubleshooting OpenArrayTM plate assembly and handling errors

Possible cause
Pipette tip was not loaded on mandrel.

Recommended action
Contact your local field service engineer for frequent occurrences (infrequent occurrences can be due to a poorly molded tip).

(continued)

OpenArrayTM plate assembly and handling errors

Observation Case leaks and bubbles inside the case
Improper sealing of the OpenArrayTM plate in the OpenArrayTM Case can lead to immersion fluid leaks or bubble formation inside the case, leading to uneven heating and imaging throughout PCR and to poor quality data. The images above are examples of OpenArrayTM plates that have been affected by immersion fluid leaks. The images show where leaked fluid has condensed on the underside of the heated cover

Possible cause Plate press was not engaged for at least 20 seconds. Damaged lid adhesive.
Damaged fill port screw gasket.
Damaged fill port screw assembly. Breaks off too easily. Oily lid or case from immersion fluid overflow.
Immersion fluid was exposed to air for too long.

Recommended action
Fully engage the plate press for at least 20 seconds.
Remove the liner and visually inspect the lid adhesives for defects. Ensure that adhesive is not damaged or warped.
Visually inspect the screw to ensure that the orange gasket is present and not damaged.
The screw may be misthreaded: unscrew it and use a new screw assembly.
Wipe off excess overflow of immersion fluid from the lid, case bottom, and crevices with 70% isopropyl alcohol, using a lint-free cloth (the cloth included with the OpenArrayTM plate is acceptable).
Do not remove the immersion fluid syringe cap or draw air bubbles into the syringe until you are ready to load.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Appendix A Troubleshooting OpenArrayTM plate assembly and handling errors

Observation windows and obscured the view of the through- holes. The best image in which to detect leaks is the s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff image. This image is taken at the start of cycling, which is where most leaks occur. See "Troubleshoot with cycling and imaging run images (QC images)" on page 41.
Sample blow-out during the addition of immersion fluid
Evaporation of reaction mixture in through-holes

Possible cause Too large of a bubble inside the OpenArrayTM Case after sealing.
Damaged plate press, leading to uneven pressure.
The reactions in A12 were compromised during the addition of immersion fluid. Injecting the immersion fluid too quickly can purge the sample out of the throughholes near the fill port. Often this is caused by the user not purging the syringe slightly before use. Too much time elapsed before the plate was sealed with lid and immersion fluid. In this example, the top half of each subarray was intentionally left open to the environment to demonstrate the effect of evaporation. "Donuts" are a result of the evaporated fluid in the though-holes.

Recommended action
Minimize the size of the bubble by tilting the OpenArrayTM Case so that the fill port is at the highest point. Slowly fill the case with immersion fluid until only a small air bubble remains. Attach the screw and wipe off any excess oil that may have spilled onto the case. Contact your field service engineer if you suspect that your plate press may be damaged. Dispense a small amount of immersion fluid onto a paper towel before use to ensure smooth operation of the syringe.
Add immersion fluid as soon as the case is removed from the plate press to minimize the likelihood of evaporation, then seal the case with the lid.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.
� Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
� Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain SDSs, see the "Documentation and Support" section in this document.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

B Appendix B Safety Chemical safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:
� Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the "Documentation and Support" section in this document.
� Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
� Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sufficient ventilation (for example, fume hood).
� Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
� Handle chemical wastes in a fume hood. � Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
� After emptying a waste container, seal it with the cap provided. � Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
� Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
� IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
AVERTISSEMENT ! PR�CAUTIONS G�N�RALES EN CAS DE MANIPULATION DE PRODUITS CHIMIQUES. Pour minimiser les risques, veiller � ce que le personnel du laboratoire lise attentivement et mette en oeuvre les consignes de s�curit� g�n�rales relatives � l'utilisation et au stockage des produits chimiques et � la gestion des d�chets qui en d�coulent, d�crites ci-dessous. Consulter �galement la FDS appropri�e pour conna�tre les pr�cautions et instructions particuli�res � respecter :
� Lire et comprendre les fiches de donn�es de s�curit� (FDS) fournies par le fabricant avant de
stocker, de manipuler ou d'utiliser les mat�riaux dangereux ou les produits chimiques. Pour obtenir les FDS, se reporter � la section � Documentation et support � du pr�sent document.
� Limiter les contacts avec les produits chimiques. Porter des �quipements de protection appropri�s
lors de la manipulation des produits chimiques (par exemple : lunettes de s�ret�, gants ou v�tements de protection).
� Limiter l'inhalation des produits chimiques. Ne pas laisser les r�cipients de produits chimiques
ouverts. Ils ne doivent �tre utilis�s qu'avec une ventilation ad�quate (par exemple, sorbonne).
� V�rifier r�guli�rement l'absence de fuite ou d'�coulement des produits chimiques. En cas de fuite
ou d'�coulement d'un produit, respecter les directives de nettoyage du fabricant recommand�es dans la FDS.
� Manipuler les d�chets chimiques dans une sorbonne.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

B Appendix B Safety
Biological hazard safety
� Veiller � utiliser des r�cipients � d�chets primaire et secondaire. (Le r�cipient primaire contient
les d�chets imm�diats, le r�cipient secondaire contient les fuites et les �coulements du r�cipient primaire. Les deux r�cipients doivent �tre compatibles avec les mat�riaux mis au rebut et conformes aux exigences locales, nationales et communautaires en mati�re de confinement des r�cipients.)
� Une fois le r�cipient � d�chets vid�, il doit �tre referm� herm�tiquement avec le couvercle fourni. � Caract�riser (par une analyse si n�cessaire) les d�chets g�n�r�s par les applications, les r�actifs et
les substrats particuliers utilis�s dans le laboratoire.
� V�rifier que les d�chets sont convenablement stock�s, transf�r�s, transport�s et �limin�s en
respectant toutes les r�glementations locales, nationales et/ou communautaires en vigueur.
� IMPORTANT ! Les mat�riaux repr�sentant un danger biologique ou radioactif exigent parfois une
manipulation sp�ciale, et des limitations peuvent s'appliquer � leur �limination.
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling warning.
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards.
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.
� U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at: https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009P.pdf
� World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

Documentation and support

Related documentation
Document
Isolation of Nucleic Acid for Respiratory Tract Microbiota Profiling Experiments Quick Reference
Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Quick Reference
TaqManTM OpenArrayTM Respiratory Tract Microbiota Plate Product Information Sheet
TaqManTM Respiratory Tract Microbiota Amplification Control Product Information Sheet
TaqManTM Universal Extraction Control Organism (B. atrophaeus) Product Information Sheet
TaqManTM Universal RNA Spike In/Reverse Transcription (Xeno) Control Product Information Sheet
QuantStudioTM 12K Flex Real�Time PCR System: OpenArrayTM Experiments User Guide
QuantStudioTM 12K Flex Real�Time PCR System Maintenance and Administration Guide
Thermo ScientificTM KingFisherTM Flex User Manual
OpenArrayTM Sample Tracker Software Quick Reference
OpenArrayTM AccuFillTM System User Guide
TaqPathTM 1-Step RT-qPCR Master Mix, CG User Guide

Publication Number MAN00018526
MAN0018529 MAN0018631 MAN0018533
MAN0018535
MAN0018534
4470935 4470689
N07669 4460657 4456986 MAN0007959

Symbols that may be displayed on the instrument, in the software, or in this guide

Symbol

Description

MANUFACTURER

CATALOG NUMBER

CONSULT INSTRUCTIONS FOR USE[1]
[1] Appendix C, "Documentation and support"

Symbol

Description

DATE OF MANUFACTURE

SERIAL NUMBER

CAUTION[1]

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

C Appendix C Documentation and support Customer and technical support
Customer and technical support
Visit thermofisher.com/support for the latest service and support information. � Worldwide contact telephone numbers � Product support information � Product FAQs � Software, patches, and updates � Training for many applications and instruments � Order and web support � Product documentation � User guides, manuals, and protocols � Certificates of Analysis � Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/ global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support.

Respiratory Tract Microbiota Profiling Experiments using OpenArrayTM Application Guide

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