Bio-Rad iScript Reverse Transcription Supermix for RT-qPCR User Manual

iScript™ Reverse Transcription Supermix for RT-qPCR

Catalog # Description

170 - 8 840 iScript Reverse Transcription Supermix for RT-qPCR, 100 µl of 5x supermix, 25 reactions
170 - 8 841 iScript Reverse Transcription Supermix for RT-qPCR, 4 x 100 µl of 5x supermix, 100 reactions

For research purposes only.

Introduction

The iScript reverse transcription supermix for RT-qPCR
(iScript RT supermix) is a sensitive, fast, and convenient
reagent for gene expression analysis using real-time reverse
transcription quantitative PCR (RT-qPCR) and standard
RT-PCR. The preblended 5x supermix contains in one tube
all the necessary components, except RNA template,
for first-strand cDNA synthesis.

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Simple and fast — short protocol time (40 min) and 1-tube
master mix format allow easy setup and fast qPCR results

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Data reproducibility — 1-tube supermix format
reduces pipetting steps and promotes consistent and
reproducible results

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Broad dynamic range — works with a broad linear
dynamic range of input total RNA (1 µg–1 pg) and
allows sensitive detection of target genes with low
expression levels

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Primer design flexibility — includes an optimum blend
of oligo(dT) and random primers to provide unbiased
representation of the 5' and 3' regions of target genes for
freedom in qPCR primer design

Storage and Stability

Store at –20°C. Guaranteed for 9 months at –20°C in a
constant temperature freezer (this reagent will not freeze
at –20°C).

Kit Contents

Reagent Description

iScript RT supermix 5x RT supermix with iScript RNase H+
(gray cap, 25 or 100 reactions) MMLV reverse transcriptase, RNase

iScript no-RT control supermix 5x no-RT control supermix formulated to
(clear cap, 50 reactions) serve as a no-enz yme control, contains all

Nuclease-free water —

inhibitor, dNTPs, oligo(dT), random primers,
buffer, MgCl

components of iScript RT supermix except
reverse transcriptase

, and stabilizers

2

Reaction Setups

Reaction Setup for a Single cDNA Synthesis Reaction

For optimal results, reactions should be assembled on ice
using appropriate reaction vessels.

Component Volume per Reaction, µl

iScript RT supermix 4
RNA template (1 µg–1 pg total RNA) Variable
Nuclease-free water Variable

Total volume 20

Reaction Setup for Multiple cDNA Synthesis Reactions

The following table shows an example of a master mix
preparation for ten reactions with 5 µl input RNA (1 µg–1 pg)
and enough excess master mix to accommodate loss during
pipetting.* For optimal results, reactions should be assembled
on ice using appropriate reaction vessels.

Component Volume per Reaction, µl

iScript RT supermix 48
Nuclease-free water 132

Total volume 180

1. Prepare the reverse transcription master mix as indicated in
the table above. Mix thoroughly by pipetting up and down
several times.

2. Add 15 µl master mix to 5 µl RNA for each reverse
transcription reaction.

3. Adjust the volume of water if the input RNA volume is not
5 µl input RNA (1 µg–1 pg), as stated in the example above.

* I f more reactions are required, scale up appropriately. The volume of supermix

provided in 25- and 100-reaction kits does not take into account the
preparation of excess master mix.

Reaction Protocol

Incubate the complete reaction mix in a thermal cycler using
the following protocol:

Priming 5 min at 25°C
Reverse transcr iption 30 min at 42°C
RT inactivation 5 min at 85°C

iScript™ Reverse Transcription Supermix for RT-qPCR

Recommendations for the Use of No-RT Control

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Interference of gene expression analysis by genomic DNA
carryover in RNA samples can be tested using the no-RT
control supermix

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Setting up a no-RT control reaction with the same amount
of total RNA as the reverse transcription reaction is
recommended to allow similar carryover of cDNA synthesis
components in qPCR for accurate detection of genomic
DNA amplicons

Recommendations for qPCR

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cDNA generated with this kit can be used directly in qPCR

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The volume of cDNA synthesis reaction used must not
exceed 10% of the qPCR volume

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cDNA can be diluted in 10 mM Tris-HCl (pH 8.0), 0.1 mM
EDTA prior to use in qPCR. The optimum cDNA dilution
must be determined based on target gene abundance and
qPCR chemistry

Related Products

Reverse transcription reagents for real-time qPCR:

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iScript advanced cDNA synthesis kit for RT-qPCR (170-8842)

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iScript cDNA synthesis kit (170-8890)

Reagents for real-time qPCR:

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SsoAdvanced™ universal SYBR® Green supermix (172-5270)

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SsoAdvanced universal probes supermix (172-5280)

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iTaq™ universal SYBR® Green supermix (172-5120)

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iTaq universal probes supermix (172-5130)

To learn more about Bio-Rad’s complete solution for
amplification, visit www.bio-rad.com/amplification.

SYBR is a trademark of Life Technologies Corporation. Bio-Rad Laboratories,
Inc. is licensed by Life Technologies Corporation to sell reagents containing
SYBR Gree n I for use in real-time PCR, for research purposes only.

Practice of the patented 5' Nuclease Process requires a lice nse from Applie d
Biosystems. The purchase of iTaq and SsoAdvanced supermixes includes an
immunity from suit under patents specified in the product insert to use only the
amount purchased for the purchaser's own internal research when used with
the separate purchase of Licensed Probe. No other patent rights are conveyed
expressly, by implication, or by estoppel. Further information on purchasing
licenses may be obtained f rom the Director of Licensing, Applied Biosystems,
850 Lincoln Centre Drive, Foster Cit y, California 94404, USA.

Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive, Hercules, CA 94547
510-741-1000

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