Instruction Manual

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.

Simple and fast — short protocol time (40 min) and 1-tube
master mix format allow easy setup and fast qPCR results

Data reproducibility — 1-tube supermix format
reduces pipetting steps and promotes consistent and
reproducible results

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

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
inhibitor, dNTPs, oligo(dT), random primers,
buffer, MgCl
2
, and stabilizers
iScript no-RT control supermix 5x no-RT control supermix formulated to
(clear cap, 50 reactions) serve as a no-enzyme control, contains all
components of iScript RT supermix except
reverse transcriptase
Nuclease-free water —
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 transcription 30 min at 42°C
RT inactivation 5 min at 85°C
Catalog # Description
170-88 40 iScript Reverse Transcription Supermix for RT-qPCR, 100 µl of 5x supermix, 25 reactions
170-88 41 iScript Reverse Transcription Supermix for RT-qPCR, 4 x 100 µl of 5x supermix, 100 reactions
iScript
™
Reverse Transcription Supermix for RT-qPCR
For research purposes only.