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 » Maxima® First Strand cDNA Synthesis Kit for RT-qPCR

Reverse Transcription

Maxima® First Strand cDNA Synthesis Kit for RT-qPCR

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Catalog#	Size, concentration	Certificate of Analysis	MSDS

K1641	for 50 react.	K1641
K1642	for 200 react.	K1642

Product information

Features

Sensitive and reproducible cDNA synthesis from a wide range of total RNA amounts (1 pg - 5 μg).
Increased synthesis rate – complete cDNA synthesis in 15 minutes.
Increased reaction temperature – up to 60°C.
Convenient format – premixed solutions for use in RT-qPCR.

Applications

Two step RT-qPCR.

Description

The Maxima® First Strand cDNA Synthesis Kit for RT-qPCR is a convenient system optimized for cDNA synthesis in two step real time quantitative RT-PCR (RT-qPCR) applications. The kit uses Maxima® Reverse Transcriptase, an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features high thermostability, robustness and increased cDNA synthesis rate compared to wild type M-MuLV RT. The Maxima® First Strand cDNA Synthesis Kit for RT-qPCR is capable of reproducible cDNA synthesis from a wide range of starting total RNA amounts (1 pg to 5 µg) at elevated temperatures (50-60°C). The synthesis reaction can be completed in 15-30 minutes.

Components of the Maxima® First Strand cDNA Synthesis Kit for RT-qPCR are premixed to save time and to reduce the possibility of pipetting errors. The contents of the kit include the Maxima® Enzyme Mix, 5X Reaction Mix and Water, nuclease-free.

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

5X Reaction Mix contains the remaining reaction components: reaction buffer, dNTPs, oligo (dT)₁₈ and random hexamer primers.

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

Quality Control

Functionally tested in two-step RT-qPCR using different starting amounts (5 ng-0.5 fg) of RNA transcript in reverse transcription reactions followed by amplification with Maxima® SYBR Green/ROX qPCR Master Mix. Efficiency is in the range of 90-110%, the slope is between -3.09 and -3.58 with a correlation coefficient >0.99.

Components

Maxima® Enzyme Mix
5X Reaction Mix
Water, nuclease-free
Detailed protocol

Figure 1. Highly sensitive two step RT-qPCR.
A – amplification plot.
B – standard curve.
Amplification of the human PPP1CA gene was performed on varying amounts of Jurkat cell total RNA (5 μg, 2.5 μg, 1 μg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg). First strand cDNA was generated with the Maxima® First Strand cDNA Synthesis Kit for RT-qPCR (#K1641). cDNA was amplified with the Maxima® Probe /ROX qPCR Master Mix (2X) using the TaqMan® assay specific for PPP1CA. Reactions were performed on an ABI 7500 Real-Time PCR instrument. 1 pg of total RNA was successfully detected. Reaction efficiency was 105%, slope-3.29, R²=0.999.

Reproducible RT-qPCR results using Maxima® First Strand cDNA Synthesis Kit for RT-qPCR

Figure 2. Reproducible RT-qPCR results using the Maxima® First Strand cDNA Synthesis Kit for RT-qPCR.
First strand cDNA was generated from 100 ng-1 pg of total Jurkat cell RNA with the Maxima® First Strand cDNA Synthesis Kit for RT-qPCR (#K1641) in 16 replicate reactions. Synthesized cDNA was used as a template in subsequent qPCR with Maxima® SYBR Green/ROX qPCR Master Mix (#K0221) on the ABI 7500 Real-Time PCR instrument. Parallel RT reactions demonstrate reproducible cDNA synthesis and low variability levels with a wide range of starting RNA amounts.

Figure 3. Greater yields and higher sensitivity using Maxima® First Strand cDNA Synthesis Kit for RT-qPCR.
Amplification of the E.coli 23S RNA gene was performed on 10-fold serial dilutions of total RNA (30 ng to 3 fg). First strand cDNA was generated using the Maxima® First Strand cDNA Synthesis Kit for RT-qPCR (red) and a reverse transcription kit from another vendor (green). cDNA was amplified using Maxima® SYBR Green/ROX qPCR Master Mix (#K0221) on the ABI 7500 Real-Time PCR instrument. All starting RNA amounts (30 ng - 3 fg) were successfully detected using the Maxima® First Strand cDNA Synthesis Kit with lower Cq and 102% reaction efficiency.

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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