Reverse transcription is a first step in two step RT-PCR. The RNA templates are reverse transcribed into cDNA which can then be amplified using PCR. The enzymes used are reverse transcriptases of different origins, most popular types are M-MuLV RT and AMV RT.

Table. Properties of reverse transcriptases.

Reverse transcriptase	Optimal reaction temperature	Active up to	Reading lenght	RNase H activity	Inactivation	Units/20 µl reaction	Amount of total RNA/20 µl reaction
Maxima® Reverse Transcriptase	50-55°C	60°C	20 kb	+	85°C, 5 min	200 u	1 pg - 5 µg
RevertAid™ Premium Reverse Transcriptase	50-55°C	60°C	20 kb	–	85°C, 5 min	200 u	1 pg - 5 µg
RevertAid™ H Minus Reverse Transcriptase	42-45°C	55°C	13 kb	–	70°C, 10 min	200 u	0.1 ng - 5 µg
RevertAid™ Reverse Transcriptase	42°C	50°C	13 kb	+	70°C, 10 min	200 u	0.1 ng - 5 µg
M-MuLV Reverse Transcriptase	37°C	37°C	9 kb	+	70°C, 10 min	40 u	100 ng - 5 µg
AMV Reverse Transcriptase	45-50°C	60°C	13 kb	++	85°C, 5 min	10 u	10 ng - 5 µg

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Products » All » PCR, qPCR, RT-PCR & dNTPs » Reverse Transcription » RevertAid™ Premium First Strand cDNA Synthesis Kit

Reverse Transcription

RevertAid™ Premium First Strand cDNA Synthesis Kit

Not available in the USA and Canada

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Catalog#	Size, concentration	Certificate of Analysis	MSDS

K1651	for 20 react.	K1651
K1652	for 100 react.	K1652

Product information

Features

High yields of full length cDNA up to 20 kb.
Efficient cDNA synthesis at wide temperature range from 45°C to 60°C.
Increased synthesis rate – complete cDNA synthesis in 30 minutes.
High sensitivity and specificity.

Applications

First Strand cDNA synthesis.
RT- PCR.
RT-qPCR.
Construction of cDNA libraries.
Generation of probes for hybridization.
RNA amplification.

Description

The RevertAid™ Premium First Strand cDNA Synthesis Kit is a complete system for highly efficient synthesis of first strand cDNA. The kit uses the RevertAid™ Premium Reverse Transcriptase (RT), an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features the highest thermostability among the derivatives of M-MuLV RT and lacks the RNase H activity. The RevertAid™ Premium First Strand cDNA Synthesis Kit allows synthesis of long cDNA up to 20 kb at elevated temperature (up to 60°C) superceeding other systems in ability to prepare full length cDNA. Due to increased synthesis rates of the enzyme first strand cDNA synthesis, the reaction can be completed in 30 min.

RevertAid™ Premium Enzyme Mix contains RevertAid™ Premium Reverse Transcriptase and RiboLock™ RNase Inhibitor. RiboLock™ RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B and C at temperatures up to 55°C.

Oligo(dT)₁₈ and random hexamer primers are supplied with the kit. Random hexamer primers bind non-specifically and are used to synthesize cDNA from all RNAs in a total RNA population. The oligo(dT)₁₈ primer selectively anneals to the 3’-end of poly(A) RNA, synthesizing cDNA only from poly(A) tailed mRNA. Gene-specific primers may also be used with the kit to prime synthesis from a specified sequence.

Water, nuclease-free is provided for reaction set-up and dilution of sample DNA. The absence of endo-, exodeoxyribonucleases, ribonucleases and phosphatases has been confirmed by appropriate quality tests.

Quality Control

Functionally tested in RT-PCR using 100 fg of control GAPDH RNA and GAPDH control primers generate a prominent 496 bp product on 1% agarose gel after ethidium bromide staining.

Components

RevertAid™ Premium Enzyme Mix
5X RT Buffer
dNTP Mix, 10 mM each
Oligo(dT)₁₈ Primer
Random Hexamer Primer
Water, nuclease-free
Detailed protocol

Figure 1. Amplification of targets up to 20 kb in two step RT-PCR.
1 µg of a total RNA from Jurkat cells (1 and 2) or a total RNA from mouse skeletal muscle (3 and 4) were used in a reverse transcription reaction with RevertAid™ Premium First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template in subsequent PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:
3.9 kb – BCL2 (human B-cell CLL/lymphoma 2)
6.8 kb – POLE (human polymerase)
13.3 kb – Dmd (mouse dystrophin)
20.2 kb – Neb (mouse nebulin)

Figure 2. Efficient cDNA synthesis at a wide temperature range.
1 µg of a total RNA from Jurkat cells were used in a reverse transcription reaction with RevertAid™ Premium First Strand cDNA Synthesis Kit using Oligo(dT)₁₈ primer at different temperatures from 45°C to 60°C. Different length amplicons (0.5 kb, 3.9 kb, 6.8 kb and 9.4 kb) were successfully amplified using Long PCR Enzyme Mix (#K0181) from the cDNA synthesized at temperatures up to 60°C.

Figure 3. High synthesis rate at 50°C using RevertAid™ Premium First Strand cDNA Synthesis Kit.
Synthesis of cDNA at 50°C for 5, 15, 30 and 60 minutes using 1µg of 7.5 kb RNA transcript as a template using RevertAid™ Premium First Strand cDNA Synthesis Kit and other kits for first strand cDNA synthesis. Reaction products were resolved on a 1% alkaline agarose gel. RevertAid™ Premium First Strand cDNA Synthesis Kit completed synthesis of 7.5 kb transcript in 5 min.

Figure 4. Higher sensitivity in two step RT-PCR using RevertAid™ Premium First Strand cDNA Synthesis Kit.
Total RNA from Jurkat cells (100 pg, 10 pg, 1 pg and 0.1pg) were used in reverse transcription reaction with RevertAid™ Premium First Strand cDNA Synthesis Kit at 50°C and other M-MuLV RT based kits at 42°C using Oligo(dT)₁₈ primer in 20 µl reaction volume. 2 µl of RT reaction mixtures were used in PCR with Maxima® Hot Start Taq DNA Polymerase (#EP0601) and primers specific for GAPDH gene.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Guidelines to Avoid RNase Contamination

RNA purity and integrity is essential for synthesis of full-length cDNA, which results in high quality RT-PCR products. Therefore, RNase contamination is always a concern when working with RNA. The RNA quality can be affected by RNase A, which is a highly stable contaminant of any laboratory environment. All components of the kit have been rigorously tested to ensure that they are RNase free. To prevent contamination both the laboratory environment and all prepared solutions must be free of RNases.

DEPC-treat all tubes and pipette tips to be used in the cDNA synthesis or use certified nuclease-free labware.
Use pipettes dedicated for RNA work.
Wear gloves when handling RNA and all reagents, as skin is an common source of RNases. Change gloves frequently.
Use certified reagents, including high quality water (e.g., nuclease-free or DEPC-treated Water).
Use an RNase inhibitor, such as RiboLock™ RNase Inhibitor, to protect template RNA.
Always assess the integrity of RNA prior to cDNA synthesis. For example, if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total RNA, the mRNA in the sample is considered to be intact.

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