Storage and stability:

SensiFAST™ cDNA Synthesis Kit SensiFAST cDNA Synthesis Kit is shipped on dry/blue ice and should be stored at -20 °C upon
receipt. When stored under optimum conditions, the reagents are stable for a minimum of one
year from date of purchase.
Shipping: On dry/blue ice Catalog numbers
Unit definitions:
BIO-65053: 50 reactions Reverse Transcriptase: One unit catalyzes the incorporation of 1 nmol of dTTP into acid-insoluble
material in 10 minutes at 37 ºC in 50 mM Tris-HCl, pH 8.6, 40 mM KCl, 1 mM MnSO4, 1 mM DTT,
Batch No.: See vial BIO-65054: 250 reactions and 0.5 mM [3H]TTP, using 200 µM oligo(dT)12-18-primed poly(A)n as template.
RNase Inhibitor: One unit inhibits 5 ng of RNase A by 50%.
Safety precautions:
Harmful if swallowed. Irritating to eyes, respiratory system and skin. Please refer to the material
Store at –20°C safety data sheet for information regarding hazards and safe handling practice.

Signal word: WARNING

Description
SensiFAST cDNA Synthesis Kit provides a rapid and very sensitive method for first strand cDNA synthesis for use in real-time PCR
studies. The 5x TransAmp™ Buffer provides highly optimized components for efficient reverse transcription, and includes a unique blend
of anchored oligo dT and random hexamer primers to ensure unbiased 3’ and 5’ coverage for enhanced data accuracy. An extremel y
efficient reverse transcriptase delivers highly robust first strand synthesis and higher cDNA yields from a wide range of input RNA
concentrations. SensiFAST cDNA Synthesis Kit offers enhanced sensitivity, efficiency and reproducibility for exceptional performance in
subsequent real-time PCR experiments.

Components
DNase I digestion of total RNA
Product Name 50 reactions 250 reactions To eliminate any residual contaminating genomic DNA that can
affect highly sensitive real-time PCR applications (e.g. probe-
5x TransAmp Buffer 200 µl 1 ml based quantification of a low abundant target), we recommend
using a high quality RNase-free DNase I during or after RNA
Reverse Transcriptase 50 µl 250 µl extraction protocols. DNase I removal by ethanol precipitation, or
with a RNA clean-up kit e.g. ISOLATE II RNA Micro Clean-Up Kit
is required before proceeding with first-strand cDNA synthesis.
SensiFAST cDNA Synthesis Mix Reaction Guidelines

Template Quality
 Intact, high-quality RNA is essential for the reverse SensiFAST cDNA Synthesis Kit Protocol
transcription reaction
 All reagents for use with RNA must be prepared using
nuclease-free, molecular biology grade water 1. Prepare the mastermix on ice.
 RiboSafe RNase Inhibitor is included in the SensiFAST cDNA 2. Vortex solutions and centrifuge briefly before use.
Synthesis Kit to help reduce template degradation and
increase yield of RT-qPCR product
 Low-copy-number genes may require an increase in starting Total RNA or mRNA (up to 1 µg) n µl
material
 Use a suitable RNA extraction reagent e.g. TRIsure™ or 5x TransAmp Buffer 4 µl
ISOLATE™ II RNA Mini Kit
Reverse Transcriptase 1 µl
RNA Priming
A unique blend of random hexamer and anchored oligo dT DNase/RNase free-water* Up to 20 µl
primers in the SensiFAST cDNA Synthesis Mix provides optimal
* Available separately (see Associated Products)
sensitivity and accuracy of first-strand cDNA synthesis. Anchored
oligo dT primers anneal precisely to the junction of the poly-A tail 3. Mix gently by pipetting.
(found on the 3’ end of most eukaryotic mRNAs) and the gene of
interest. This ensures that the coding 3’ end of mRNAs are 4. Set up the following program in a thermal cycler:
always represented. The reverse transcriptase can also prime  25 °C for 10 min (primer annealing)
from the random hexamers, to give broad coverage of all the
 42 °C for 15 min (reverse transcription)
regions of the RNA and thus a cDNA pool representative of the
transcritpome. The combined benefits of both priming strategies  Optional Step: 48 °C for 15 min (for highly-structured RNA)
offers enhanced data quality.  85 °C for 5 min (inactivation)
 4 °C hold (or chill on ice)
Reverse Transcription
Efficient reverse transcription can normally be achieved at 42 °C 5. Use up to 4 µl (1/5th volume) of cDNA synthesis reaction product in a
for 15 minutes, as the TransAmp Buffer contains reverse 20 µl volume real-time PCR. If desired, reaction product can be diluted
transcriptase enhancers that reduce complex RNA secondary in 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA prior to use.
structure. For templates that have a high degree of structure,
6. Alternatively, store reaction product or diluted cDNA at 4 °C for 1 week
such as viral RNA and some plant RNA, we suggest using an or -20 °C for long term storage.
additional 15 minute 48 °C incubation step.

No RT Control
It is important to always include the appropriate ‘no RT’ or ‘minus This protocol is intended for use as a guide only; conditions will vary from
RT’ control reactions in your experimental design. As the reverse reaction to reaction and may need optimization.
transcriptase is a separate component of the SensiFAST cDNA
Synthesis Kit, it is possible to include a formal cDNA synthesis
control that includes all components except the reverse
transcriptase.

PI-50476 V6 Website: www.bioline.com/ email: info@bioline.com

 Troubleshooting

Problem Possible Cause Recommendation

Analyze RNA on a denaturing gel to verify integrity.

RNA degraded
Ensure that all reagents are RNase-free.

Remove inhibitors, such as SDS, EDTA, formamide and pyrophosphate, by ethanol

RNA contained an RT inhibitor
precipitation of RNA, including a 70% ethanol wash step.
No cDNA synthesis

Increase the primer annealing step from 10 min up to 15 min. Increase the reverse transcription
Reaction conditions not optimal
step from 15 min up to 30 min.

Increase the amount of starting RNA, this can be an important factor when amplifying low-copy
Not enough starting RNA
genes from total RNA.

Non-specific annealing of Increase the annealing temperature in real-time PCR.

primers to template Check for presence of pseudogenes.

Poor specificity in
real-time PCR Primer dimers Redesign primers to prevent self-annealing. Set up reactions on ice.

Treat RNA with DNase I and re-purify. If possible, use intron-spanning primers in real-time
Genomic DNA contamination
PCR.

Product in RNA contaminated with

Treat samples with DNase I and re-purify. Use intron-spanning primers if possible.
‘no RT’ control genomic DNA

Technical Support: Associated products:

If the troubleshooting guide does not solve the difficulty you are Product Name Cat. No.
experiencing, please contact Technical Support with details of
reaction setup, cycling conditions and relevant data. SensiFAST™ SYBR No-ROX Kit BIO-98002

Email: tech@bioline.com
SensiFAST™ Probe No-ROX Kit BIO-86005

ISOLATE™ II RNA Mini Kit BIO-52072

ISOLATE™ II RNA Micro Kit BIO-52075

TRIsure™ BIO-38032

HyperLadder™ 1kb BIO-33025

Trademarks
SensiFAST™, TransAmp™, ISOLATE™, TRIsure™ and HyperLadder™
are trademarks of Bioline Reagents Ltd.

____________________________________________________________________________________________________________________________
Bioline Reagents Ltd Bioline USA Inc. Bioline GmbH Bioline (Aust) Pty. Ltd Bioline France Meridian Bioscience Asia Pte Ltd
UNITED KINGDOM USA GERMANY AUSTRALIA FRANCE SINGAPORE

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PI-50476 V6 Website: www.bioline.com/ email: info@bioline.com