Applied Biosystems 7900HT User manual

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Applied Biosystems 7900HT
Micro Fluidic Card
Getting Started Guide
FINAL DRAFT June 5, 2003
Please return to Clarissa Kao (x6673) MS 458 by June 11.

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© Copyright 2003, Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Notice to Purchaser: Disclaimer of License
The Micro Fluidic Card is optimized for use in the Polymerase Chain Reaction (PCR) and 5' nuclease detection methods covered
by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No license under these patents to use the
PCR process or 5' nuclease detection methods is conveyed expressly or by implication to the purchaser by the purchase of this
product. A license to use the PCR process for certain research and development activities accompanies the purchase of certain
Applied Biosystems reagents when used in conjunction with an authorized thermal cycler, or is available from Applied
Biosystems. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of
Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc.,
1145 Atlantic Avenue, Alameda, California 94501.
Notice to purchaser: Limited License
TaqMan® probes are covered by U.S. Patent 5,723,591 and foreign counterparts and patents pending owned by Applera
Corporation, and are covered by U.S. Patents 5,801,155 and 6,084,102 and foreign counterparts licensed to Applied Biosystems.
Micro Fluidic Card developed in collaboration with 3M Company.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that
may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall
Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use
of this document.
Applied Biosystems and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain
other countries.
AB (Design), ABI PRISM, Applera, and Assays-On-Demand are trademarks of Applera Corporation or its subsidiaries in the U.S.
and/or certain other countries.
AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
All other trademarks are the sole property of their respective owners.
Part Number 4319399 Rev. C
7/2003

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Contents
Applied Biosystems Micro Fluidic Card Getting Started Guide iii
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Safety Alert Words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Chemical Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Chemical Safety Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
About MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Obtaining MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Site Preparation and Safety Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
How to Obtain Services and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
About the Getting Started Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
About the Micro Fluidic Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
About Micro Fluidic Card Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . 5
Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Process Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Components and Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . 7
Ordering Additional Cards or Reagents . . . . . . . . . . . . . . . . . . . . . . . 8
Items Required But Not Supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Notes About Micro Fluidic Card Experiments . . . . . . . . . . . . . . . . . . . . . 12
About the Comparative CTMethod of Relative Quantification . . . . . 12
Significance of the Calibrator Sample . . . . . . . . . . . . . . . . . . . . . . . 12
Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Performing Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Where You Are in the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Guidelines for Preparing RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Starting Amount of Total RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Storing cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Preparing and Running the Micro Fluidic Cards (PCR) . . . . . . . . . . . . . . 15
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Where You Are in the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Process Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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iv Applied Biosystems Micro Fluidic Card Getting Started Guide
Guidelines for Preparing the PCR Mix . . . . . . . . . . . . . . . . . . . . . . . 17
Amount of cDNA in the PCR Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Preparing the Sample-Specific PCR Mix . . . . . . . . . . . . . . . . . . . . . 17
Guidelines for Loading Cards and Operating the Centrifuge . . . . . 19
Creating Micro Fluidic Card Documents . . . . . . . . . . . . . . . . . . . . . 20
Performing the Micro Fluidic Card Run . . . . . . . . . . . . . . . . . . . . . . 26
Performing Real-Time Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Where You Are in the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Absolute vs. Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . 30
Additional Tasks Related to Micro Fluidic Card Use . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Appendix A Micro Fluidic Card Information CD . . . . . . . . . . . . . . . . . 33
About the Micro Fluidic Card Information CD . . . . . . . . . . . . . . . . . 33
Assay Information File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Card Map Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
SDS Setup File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

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Safety
Applied Biosystems Micro Fluidic Card Getting Started Guide 1
Safety
Safety Alert
Words
Four safety alert words appear in Applied Biosystems user
documentation. Each word implies a particular level of observation
or action, as described below:
IMPORTANT! Indicates information that is necessary for proper
instrument operation, accurate chemistry kit use, or safe use of a
chemical.
Indicates a potentially hazardous situation that, if
not avoided, can result in minor or moderate injury. It can also alert
against unsafe practices, damage to an instrument, or loss of data.
Indicates a potentially hazardous situation that, if
not avoided, can result in serious injury or death.
Indicates an imminently hazardous situation that,
if not avoided, will result in serious injury or death. This signal word
is to be limited to the most extreme situations.
Chemical Hazard
Warning
CHEMICAL HAZARD. Some of the chemicals
used with Applied Biosystems instruments and protocols are
potentially hazardous and can cause injury, illness, or death.
Chemical Safety
Guidelines
To minimize the hazards of chemicals:
• Read and understand the MSDSs provided by the chemical
manufacturer before you store, handle, or work with any
chemicals or hazardous materials. See “About MSDSs.”
• Minimize contact with chemicals. When handling chemicals,
wear appropriate personal protective equipment such as safety
glasses, gloves, and protective clothing. For additional safety
guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical
containers open. Use only with adequate ventilation (for
example, a fume hood). For additional safety guidelines, consult
the MSDS.

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2 Applied Biosystems Micro Fluidic Card Getting Started Guide
• Check regularly for chemical leaks or spills. If a leak or spill
occurs, follow the cleanup procedures recommended in the
MSDS.
• Comply with all local, state/provincial, or national laws and
regulations related to chemical storage, handling, and disposal.
About MSDSs Chemical manufacturers supply current Material Safety Data Sheets
(MSDSs) with shipments of hazardous chemicals to new customers.
They also provide MSDSs with the first shipment of a hazardous
chemical to a customer after an MSDS has been updated. MSDSs
provide the safety information you need to store, handle, transport,
and dispose of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous
chemical, be sure to replace the appropriate MSDS in your files.
Obtaining MSDSs You can obtain from Applied Biosystems the MSDS for any chemical
supplied by Applied Biosystems. This service is free and available 24
hours a day.
To obtain MSDSs:
1. Go to https://docs.appliedbiosystems.com/msdssearch.html.
2. In the Search field, type in the chemical name, part number, or
other information that appears in the MSDS of interest. Select
the language of your choice, then click Search.
3. Find the document of interest, right-click the document title,
then select any of the following:
•Open – To view the document
•Print Targe t – To print the document
•Save Target As – To download a PDF version of the
document to a destination that you choose
4. To have a copy of a document sent by fax or e-mail, select Fax
or Email to the left of the document title in the Search Results
page, then click RETRIEVE DOCUMENTS at the end of the
document list.
5. After you enter the required information, click View/Deliver
Selected Documents Now.

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How to Obtain Services and Support
Applied Biosystems Micro Fluidic Card Getting Started Guide 3
Site Preparation
and Safety Guide
A site preparation and safety guide is a separate document sent to all
customers who have purchased an Applied Biosystems instrument.
Refer to the guide written for your instrument for information on site
preparation, instrument safety, chemical safety, and waste profiles.
Waste profiles help you plan for the handling and disposal of waste
generated by operation of the instrument. Read the waste profiles and
all applicable MSDSs for your instrument before handling or
disposing of chemical waste.
Waste Disposal If potentially hazardous waste is generated when you operate the
instrument, you must:
• Characterize (by analysis if necessary) the waste generated by the
particular applications, reagents, and substrates used in your
laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported,
and disposed of according to all local, state/provincial, and/or
national regulations.
Note: Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.
How to Obtain Services and Support
For the latest services and support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for Services
and Support.
At the Services and Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates
of analysis, and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Services and Support page provides access to
worldwide telephone and fax numbers to contact Applied Biosystems
Technical Support and Sales facilities.

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4 Applied Biosystems Micro Fluidic Card Getting Started Guide
Introduction
About the Getting
Started Guide
This guide accompanies the Micro Fluidic Card. It provides
instructions for the preparation, execution, and analysis of Micro
Fluidic Card experiments.
If you are familiar with the theory behind the Applied Biosystems
7900HT Micro Fluidic Card (Micro Fluidic Card) chemistry or the
ABI PRISM®7900HT Sequence Detection System (7900HT system)
data collection, the information in this document serves as a quick
reference guide for conducting experiments using the Micro Fluidic
Card.
If this is the first time you are using the Micro Fluidic Card, this
guide refers you to the appropriate sections of the ABI PRISM®
7900HT Sequence Detection Systems and SDS Enterprise Database
User Guide (PN 4317596, referred to as the user guide for the
7900HT system throughout the rest of this document) for more
information.
Note: A copy of the user guide is shipped with the 7900HT system.
An electronic copy is included on the Micro Fluidic Card Information
CD that comes with your Micro Fluidic Card order.
About the Micro
Fluidic Card
The Micro Fluidic Card allows you to use the 7900HT system for
profiling gene expression using the Comparative CTMethod of
relative quantification. The card evaluates from one to eight cDNA
samples or controls generated from total RNA in a two-step RT-PCR
experiment.
Key Features
Following are the key features of the Applied Biosystems 7900HT
Micro Fluid Card:
• Assays-on-Demand™Gene Expression Products for all real-time
RT-PCR applications
• Flexible card format handles multiple sample/target
combinations
• Small-volume design minimizes sample and reagent
consumption
• Streamlined reaction set-up saves time and reduces labor-
intensive steps

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Introduction
Applied Biosystems Micro Fluidic Card Getting Started Guide 5
• Provides access to high-throughput, 384-well format without
liquid-handling robotics
• Micro Fluidic Cards, when used with the 7900HT system, detect
2-fold discrimination at the 99.7% confidence level
More Information About Micro Fluidic Cards
Chapter 4 of the user guide for the 7900HT system contains more
information about the Micro Fluidic Card, including
• How it works – The Micro Fluidic Card functions as an array of
reaction vessels for the PCR/sequence detection step.
• Card components – Each card is a specially developed 384-well
consumable divided into eight sets of assays.
• Internal Structure – Micro Fluidic Cards have fill reservoirs, or
ports, into which you load your samples.
• Detectors – Micro Fluidic Cards permit the amplification of the
endogenous control and targets in cDNA samples using
fluorogenic 5´ nuclease assays.
Note: Real-time PCR in Micro Fluidic Cards are run as singleplex
reactions.
About Micro
Fluidic Card
Chemistry
Micro Fluidic Card experiments use a two-step RT-PCR process. In
the reverse-transcription (RT) step, cDNA is reverse transcribed from
total RNA samples using random primers from the High Capacity
cDNA Archive Kit. Additional details about the RT-PCR process are
contained in the High Capacity cDNA Archive Kit Protocol
(PN 4322169).
In the PCR step, PCR products are synthesized from cDNA samples
using the TaqMan Universal PCR Master Mix. The PCR step
employs the 5´ nuclease assay, which is described in Appendix C of
the user guide for the 7900HT system.
Preventing
Contamination
PCR assays require special laboratory practices to avoid false
positive amplifications (Kwok and Higuchi, 1989). The high
throughput and repetition of these assays can lead to amplification of
a single DNA molecule (Saiki et al., 1985; Mullis and Faloona,
1987).
For more information about preventing contamination, refer to
Chapter 4 of the user guide for the 7900HT system.

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6 Applied Biosystems Micro Fluidic Card Getting Started Guide
Process
Flowchart
The following diagram provides an overview of the Micro Fluidic
Card process.
Performing Reverse
Tra nscription
(cDNA generation)
Interpreting Results
Performing Real-Time Data
Analysis
(7900HT system and SDS
software)
Preparing and Running the
Micro Fluidic Cards (PCR)
See “Performing Reverse
Transcription” on page 13.
See “Preparing and
Running the Micro Fluidic
Cards (PCR)” on page 15.
See “Performing Real-
Time Data Analysis” on
page 29.
See Chapter 6 of the user
guide for the 7900 HT

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Materials and Equipment
Applied Biosystems Micro Fluidic Card Getting Started Guide 7
Materials and Equipment
Components and
Storage
Conditions
The Micro Fluidic Card Upgrade consists of two kits, the 7900HT
Micro Fluidic Card Hardware Upgrade Kit (PN 4329012) and the
7900HT Micro Fluidic Card Chemical Installation Kit (PN 4340090).
The following tables list the components of each kit and, where
applicable, their storage conditions. The tables also list the part
numbers for kit components that are available for individual
purchase.
All Micro Fluidic Cards ship at ambient temperature. Store Micro
Fluidic Cards at the recommended temperature upon receipt.
7900HT Micro Fluidic Card Hardware Upgrade Kit (PN 4329012)
7900HT Micro Fluidic Card Chemical Installation Kit (PN 4340090)
Component Part Number
Applied Biosystems 7900HT Micro Fluidic Card Getting
Started Guide
4319399
Micro Fluidic Card Sample Block
(Also referred to as the thermal cycler module or thermal
block)
4329011
Micro Fluidic Card Sealer –
Sorvall/Heraeus Custom Buckets and Adaptors (4 pcs.) –
7900HT Heated Cover –
SDS 2.1 Software CD –
Component Part Number Storage
Calibration Micro Fluidic Card (2 pcs.) – ambient
Installation Micro Fluidic Card (12 pcs.)
(Also referred to as the TGF-β
Instrument Verification Card)
– 2–8 °C
Spectral Calibration Kit, 96-Well 4328639 -15 to -25 °C

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8 Applied Biosystems Micro Fluidic Card Getting Started Guide
Ordering
Additional Cards
or Reagents
To order additional Micro Fluidic Cards or reagents, visit the Applied
Biosystems Web site at http://www.appliedbiosystems.com.
Micro Fluidic Cards are available in one of ten configurations:
Spectral Calibration Kit, 384-Well 4323977 -15 to -25 °C
Spectral Calibration Reagent Kit – -15 to -25 °C
Ta q M a n ®Human Raji cDNA (25ng/µL) – -15 to -25 °C
Ta q M a n ®Universal PCR Master Mix 4304437 2–8 °C
Component Part Number Storage
Description Part
Number
Total
Number
of Targets
Number
of
Samples
Number of Assay
Replicates Per
Port
7900HT Gene Expression Micro
Fluidic Card Configuration 1
4342247 12 8 4
7900HT Gene Expression Micro
Fluidic Card Configuration 2
4342249 24 8 2
7900HT Gene Expression Micro
Fluidic Card Configuration 3
4342251 24 4 4
7900HT Gene Expression Micro
Fluidic Card Configuration 4
4342253 48 8 1
7900HT Gene Expression Micro
Fluidic Card Configuration 5
4342255 48 4 2
7900HT Gene Expression Micro
Fluidic Card Configuration 6
4342257 48 2 4
7900HT Gene Expression Micro
Fluidic Card Configuration 7
4342259 96 4 1
7900HT Gene Expression Micro
Fluidic Card Configuration 8
4342261 96 2 2

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Materials and Equipment
Applied Biosystems Micro Fluidic Card Getting Started Guide 9
The 7900HT Immune Profiling Micro Fluidic Card (PN 4342510),
which facilitates the analysis of gene expression targets known to
have implications in immune response, is also available.
Items Required
But Not Supplied
In addition to the components supplied with the Micro Fluidic Card,
other items are required for this protocol. As noted below, some of
the items listed are available from major laboratory suppliers (MLS).
Note: You need only one of the eight centrifuges and one of the two
rotors listed in the table.
Instruments and Accessories
7900HT Gene Expression Micro
Fluidic Card Configuration 9
4342263 96 1 4
7900HT Gene Expression Micro
Fluidic Card Configuration 10
4342265 380 1 1
Description Part
Number
Total
Number
of Targets
Number
of
Samples
Number of Assay
Replicates Per
Port
Item Source Part
Number
ABI PRISM®7900HT
Sequence Detection System
Applied
Biosystems
Contact
Applied
Biosystems
Applied Biosystems 7900HT Micro
Fluidic Cards
Applied
Biosystems
Contact
Applied
Biosystems
High Capacity cDNA Archive Kit Applied
Biosystems
4322171
Ta q M a n ®Universal PCR Master Mix aApplied
Biosystems
4304437
Microcentrifuge MLS ---

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10 Applied Biosystems Micro Fluidic Card Getting Started Guide
One of the following centrifuges:b,c
Sorvall®Legend T Centrifuge
(EASYSet)
nonrefrigerated tabletop centrifuge
Kendro
Fisher Scientific
75004367
Sorvall®Legend T (QUIKSet)
nonrefrigerated tabletop centrifuge
Kendro
Fisher Scientific
75004366
Heraeus Multifuge 3S (EASYSet)
nonrefrigerated tabletop centrifuge
Cardinal Health 75004365
Heraeus Multifuge 3L (QUIKSet)
nonrefrigerated tabletop centrifuge
Cardinal Health 75004364
Sorvall®Legend RT (EASYSet)
refrigerated tabletop centrifuge
Kendro
Fisher Scientific
75004377
Sorvall®Legend RT (QUIKSet)
refrigerated tabletop centrifuge
Kendro
Fisher Scientific
75004376
Heraeus Multifuge 3SR (EASYSet)
refrigerated tabletop centrifuge
Cardinal Health 75004375
Heraeus Multifuge 3LR (QUIKSet)
refrigerated tabletop centrifuge
Cardinal Health 75004374
One of the following rotors:d
4-Place Swinging Bucket Rotor
Requires tool (included) for securing
to centrifuge.
Kendro
Fisher Scientific
Cardinal Health
75006445
4-Place Swinging Bucket Rotor
Twist-on fixture. Does not require tool
for securing to centrifuge.
Kendro
Fisher Scientific
Cardinal Health
75006434
a.The TaqMan Universal PCR Master Mix (5 mL) is 2X in concentration and
contains sufficient reagent for approximately 12 Micro Fluidic Cards. The
mix is optimized for TaqMan reactions and contains AmpliTaq Gold®DNA
Polymerase, AmpErase®UNG, dNTPs with dUTP, Passive Reference, and
optimized buffer components.
b. All centrifuges have digital displays. The user interface either has a
membrane touchpad (for EASYSet models) or is knob-activated (for
QUIKSet models). Rotors and other accessories are not included with the
centrifuge.
c. The centrifuge part numbers provided in the table are for North America
only.
d. Rotors do not include buckets. Both rotors accept the Sorvall/Heraeus
Custom Buckets and Adaptors (4 pcs.) that come with the 7900HT Micro
Fluidic Card Hardware Upgrade Kit (PN 4329012).

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Materials and Equipment
Applied Biosystems Micro Fluidic Card Getting Started Guide 11
Lab Equipment and Materials
Item Source Part
Number
MicroAmp®Reaction Tubes with Caps,
0.2-mL
Applied
Biosystems
N8010612
Rainin F-100 micropipette (fixed 100-µL
volume)a
a. Applied Biosystems recommends Rainin pipettes and tips; however, you
may determine that other brands are equivalent.
Rainin F-100
Rainin Fine Point pipette tips (100-µL)aRainin HR-100F
Gloves, disposable, powder-free MLS ---
Microcentrifuge tubes, sterile 1.5-mL MLS ---
Water, RNase/DNase-free, deionized MLS ---
Scissors --- ---

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12 Applied Biosystems Micro Fluidic Card Getting Started Guide
Notes About Micro Fluidic Card Experiments
About the
Comparative CT
Method of
Relative
Quantification
Relative gene expression values can be obtained from
7900HT system data using the Comparative CTMethod for relative
quantification. In the Comparative CTMethod, quantity is expressed
relative to a calibrator sample that is used as the basis for comparative
results. Therefore, the calibrator is the 1✕sample and all other
quantities are expressed as an n-fold difference relative to the
calibrator.
Note: For more information on the Comparative CTMethod for
relative quantification, refer to Livak & Schmittgen, 2001 and the
ABI PRISM®7700 Sequence Detection System User Bulletin #2:
Relative Quantitation of Gene Expression (PN 4303859).
Significance of
the Calibrator
Sample
All Micro Fluidic Card relative quantification experiments require
data from a calibrator sample. During analysis, the ABI PRISM®
7900HT Sequence Detection System software (SDS 2.1 software)
calculates the levels of target gene expression in samples relative to
the level of expression in the calibrator. Thus, the calibrator sample is
an integral part of the relative quantification calculation because it
serves as the basis for the comparative results.
Examples of possible calibrator samples include:
• A zero timepoint sample in a time-course experiment
• An untreated sample (versus treated samples)
• A resting sample (versus activated samples)
Design
Guidelines
The most important factor to consider when designing Micro Fluidic
Card experiments is how you intend to analyze your data:
• If you want to analyze your data using Applied Biosystems RQ
Manager Software, set up relative quantification plate
documents. (This is the recommended method.)
• If you want to analyze your data using a custom application, set
up absolute quantification plate documents.
IMPORTANT! Once you have set up a relative quantification
document, you cannot convert it into an absolute quantification
document. Similarly, you cannot convert an absolute quantification
document into a relative quantification document. (For more
information, see Chapter 6 of the user guide for the 7900HT system.)

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Performing Reverse Transcription
Applied Biosystems Micro Fluidic Card Getting Started Guide 13
Performing Reverse Transcription
Summary Synthesis of cDNA from total RNA samples using the High Capacity
cDNA Archive Kit (PN 4322171) is the first step in the two-step
RT-PCR gene expression quantification experiment.
Use the manual method for converting total RNA into cDNA, as
specified in the High Capacity cDNA Archive Kit Protocol
(PN 4322169).
Note: The protocol is not shipped with the High Capacity cDNA
Archive Kit. You must download the protocol from the
Applied Biosystems Documents on Demand Web site at
http://docs.appliedbiosystems.com/search.taf
To search for the document, select ABI PRISM™6100 Nucleic Acid
PrepStation under Products and click Search. The protocol is listed
under the Protocols heading.
Where You Are in
the Procedure
You
Are Here
1. Prepare total RNA
samples.
2. Use the manual method of
the High Capacity cDNA
Archive Kit to generate
cDNA.
Performing Reverse
Transcript ion
(cDNA generation)
Performing Reverse Transcription
(cDNA generation)
Performing Real-Time Data Analysis
(7900HT system and SDS software)
Preparing and Running the Micro
Fluidic Cards (PCR)
Interpreting Results

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14 Applied Biosystems Micro Fluidic Card Getting Started Guide
Guidelines for
Preparing RNA
Use only total RNA samples to generate cDNA for use with the
Micro Fluidic Card. The 18S RNA endogenous control assay cannot
accurately evaluate cDNA generated from poly A+RNA samples
because most of the rRNA has been removed from them.
IMPORTANT! See the Applied Biosystems product insert included
with your reagent kit for specific information on the recommended
template for use with the kit.
The High Capacity cDNA Archive Kit Protocol (PN 4322169) also
contains additional guidelines for preparing the RNA template.
Starting Amount
of Total RNA
The starting amount of total RNA that you need to convert to cDNA
depends on which Micro Fluidic Card Configuration you intend to
use. For example, Configuration 1 allows you to load eight different
samples into the eight fill reservoirs on the card, whereas
Configuration 10 requires you to load the same sample into all eight
fill reservoirs on the card. Refer to the assay information file (AIF)
on the Micro Fluidic Card Information CD to confirm the number of
fill reservoirs per sample.
Based on guidelines for Micro Fluidic Cards (discussed in “Amount
of cDNA in the PCR Mix” on page 17), you should load between
1–100 ng of total RNA converted to cDNA per fill reservoir.
Storing cDNA After thermal cycling, store all cDNA samples at -15 to -25 °C. To
minimize repeated freeze-thaw cycles of cDNA, Applied Biosystems
recommends that you store your cDNA samples in aliquots.

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Preparing and Running the Micro Fluidic Cards (PCR)
Applied Biosystems Micro Fluidic Card Getting Started Guide 15
Preparing and Running the Micro Fluidic Cards
(PCR)
Summary Amplification of cDNA is the second step in the two-step RT-PCR
experiment. In this step, the sample-specific PCR mix is loaded into a
Micro Fluidic Card. The Micro Fluidic Card is run on the 7900HT
system, where AmpliTaq Gold®DNA polymerase amplifies the
cDNA synthesized from the original total RNA sample.
Where You Are in
the Procedure
You
Are Here
1. Prepare the cDNA samples
for the real-time PCR run
(sample-specific PCR mix).
2. Pipette the sample-
specific PCR mix into a
Micro Fluidic Card.
3. Centrifuge the Micro
Fluidic Card.
4. Seal the Micro Fluidic Card
and trim-off fill reservoirs.
5. Create a Micro Fluidic
Card document.
– If you are using SDS
2.1 Software to analyze
results, create a relative
quantification
document.
– If you are using custom
software to analyze
results, create an
absolute quantification
document.
6. Perform the run.
Loading, Centrifuging,
Sealing, Trimming, and
Running the Micro Fluidic
Cards
Performing Reverse Transcription
(cDNA generation)
Performing Real-Time Data Analysis
(7900HT system and SDS software)
Preparing and Running the Micro
Fluidic Cards (PCR)
Interpreting Results

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16 Applied Biosystems Micro Fluidic Card Getting Started Guide
Process
Overview
The PCR process consists of the following procedures:
1. Preparing the sample-specific PCR mix.
See page 17.
2. Loading the Micro Fluidic Cards.
See Chapter 4 of the user guide for the 7900HT system.
3. Centrifuging the Micro Fluidic Cards.
See Chapter 4 of the user guide for the 7900HT system and
page 19 for Micro Fluidic Card-specific guidelines on
centrifuge operation.
4. Sealing the Micro Fluidic Cards and trimming-off fill
reservoirs.
See Chapter 4 of the user guide for the 7900HT system.
IMPORTANT! The sealing process is unidirectional. Once
you go over the card in one direction, you cannot reverse the
process.
Note: Sealing tape is not required for the card sealer. The
sealer makes grooves into the foil side of the card, sectioning
off the sample pocket from the main channel.
5. Creating Micro Fluidic Card Documents.
See page 20.
6. Performing Micro Fluidic Card Runs.
See page 26.