Thermo Fisher Scientific OpenArray Quick Start Guide
QUICK REFERENCE
Respiratory Tract Microbiota P
rofiling Experiments v2 using
OpenArray™ plates
Pub. No. MAN0019509 Rev. A.0
Note: For safety and biohazard guidelines, see the “Safety”
appendix in the Respiratory Tract Microbiota Profiling Experiments
v2 using OpenArray™ Application Guide (Pub. No. MAN0019506).
Read the Safety Data Sheets (SDSs) and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
This document is intended as a benchtop reference for
experienced users. See “Related documentation” on page 5 for
resources that contain detailed instructions and troubleshooting.
Perform preamplification and dilute samples
Perform preamplification
Note: Preamplification of the TrueMark™ Respiratory Panel 2.0
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.
4. Gently vor
tex 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 Step Temperature Time
Hold
Hold
Hold Activation 95°C 2 minutes
Cycling (14
cycles)
Hold Inactivation 99.9°C 10 minutes
Hold — 4°C Hold
[1]
eat-labile UNG is completely inactivated during the initial ramp to 95°C.
H
UNG
incubation
Reverse
anscription
tr
Denaturation 95°C 15 seconds
Annealing/
Extension
[1]
25°C 2 minutes
50°C 30 minutes
60°C 2 minutes
Component
TaqPath™ 1-Step RT-qPCR Master Mix,
CG
TrueMark™ Respiratory Panel 2.0 PreAmp
[1]
Primers
[1]
T
he TrueMark™ Respiratory Panel 2.0 PreAmp Primers contains primers
for all respiratory tract microbiota assays plus the TaqMan™ Universal RNA
Spike In/Reverse Transcription (Xeno) Control and the RNase P RPPH1
gene, but does not contain primers for the TaqMan™ Universal Extraction
Control Organism (B. atrophaeus).
Volume per
r
eaction
2.5 µL
2.5 µL
2. Distribute PreAmp Reaction Mix, then Nuclease-free Water
or sample nucleic acid to the appropriate wells of a 96-well
plate.
No-template
Component Sample reaction
PreAmp Reaction
Mix
Sample DNA or
NEC
Nuclease-free Water — 5 µL
Total volume per
reaction
5 µL 5 µL
5 µL —
10 µL 10 µL
contr
reaction
ol (NTC)
6. Store the plate on ice until dilution for PCR (see “Dilute the
preamplified sample” on page 1).
Dilute the preamplified sample
To determine dilution volumes, first determine the total volume
of diluted preamplified sample that is required for PCR (see
“Prepare and run respiratory tract microbiota profiling experiments
with OpenArray™ plates” on page 2). 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.
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 µL).
b. Add the appropriate volume of Nuclease-free Water to
each sample and control well (for example, 18 µL).
4. Seal the plate with new adhesive film.
3. Seal the plat
e with adhesive film.
For Research Use Only. Not for use in diagnostic procedures.
5. Vortex the plate for 10 seconds, then briefly centrifuge.
b. Select the folders indicat
ed in this table:
6. Proceed directly to PCR (see “Prepare and run respiratory
tract microbiota profiling experiments with OpenArray
™
plates” on page 2 ).
Seal the plate that contains the unused portion of the undiluted
preamplified samples, then store at –20°C.
Prepare and run respiratory tract microbiota
profiling experiments with OpenArray™ plates
One-time procedures
Set up default folders and software preferences
Set up the default file locations and preferences before using
the OpenArray™ AccuFill™ 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 AccuFill™ software:
• TPF Files
• Sample Tracker 96-well Input
• Sample Tracker 384-well CSV Files
• Loaded TPF Files
2. (Optional) Copy a template file into the OpenArray™ Sample
Tracker Software folder.
• Navigate to this folder on your
computer: <drive>:\Program Files
(x86)\Applied Biosystems\OpenArray Sample
Tracker\examples.
• Copy the 96‑Well Sample Plate 1.csv template
file, which is provided with the OpenArray™ Sample
Tracker Software.
• Paste the template file into the Sample Tracker 96‑well
Input folder.
AccuFill
OpenArray
Plate File Input
Folder
Sample Plate
File Folder
Loaded
OpenArray
Plate File
Folder
5. In the QuantS
™
folder
™
™
Default folder Folder contents
TPF Files TPF files for the
Sample Tracker
384-well CSV Files
Loaded TPF Files Integrated TPF files
OpenArray™ plates,
with assay name
and location
CSV 384-well
sample plate layout
files
generated during
processing with the
AccuFill™ software.
tudio™ 12K Flex Software, select Tools4
Preferences4OpenArray, then select the Loaded TPF
Files folder for the software Setup Folder.
Note: If the QuantStudio™ 12K Flex Software is not on
the same computer as the AccuFill™ software, transfer the
loaded TPF files to the computer running the QuantStudio
™
12K Flex Software.
Obtain TPF files
Go to thermofisher.com/OA-platefiles to obtain the TPF files for
the TrueMark™ Respiratory Panel 2.0, OpenArray™ Plate.
Set up the optimized folder locations and software preferences
before downloading TPF files to your computer.
3. In the OpenArray™ Sample Tracker Software, select View4
Preferences, then enter the following preferences:
Field Selection
Experiment Type Gene Expression
OpenArray™ Plat
Pipettor Fixed or Adjustable tip spacing
Import Data Directory Sample Tracker 96-well Input
Export Data Directory Sample Tracker 384-well CSV Files
4. In the AccuFill™ softwar
e Select the OpenArray™ format that
will be run most often, such as
Gene Expression – 112.
e, select Instrument4Edit
Preferences, then:
a. Select Require Sample Integration.
2 Respir
atory Tract Microbiota Profiling Experiments v2 using OpenArray™ Quick Reference
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