PCR/Amplification

Reverse Transcriptase PCR (RT-PCR)

RT-PCR Background and Reagents


 What is RT-PCR?

Reverse transcriptase PCR, or RT-PCR, is a variation of standard PCR technique, which involves the amplification of specific mRNA obtained from very small samples. It eliminates the need for the tedious mRNA purification process required for conventional cloning techniques. In RT-PCR, reverse transcriptase and an RNA sample are used in addition to the standard PCR reagents. The reaction mixture is heated to 37 ˚C, which allows for the production of cDNA copy from the RNA sample by reverse transcriptase. This cDNA then anneals to one of the primers leading to first strand synthesis. Standard PCR follows from here. Ultimately, dsDNA is produced.

As always, the primers used must match the project. If amplification of all of a cell’s mRNA is required, oligo (dT) primers are sufficient because they anneal to the poly A tails. If a specific mRNA is to be amplified, a coding region-specific primer can be used.

RT-PCR is a common diagnostic tool in microbiology and virology. This technique is frequently combined with real time PCR (qPCR), which is widely used for the quantification of transcript levels in cells and tissues.1

Low abundance or difficult secondary structure of these transcripts may exceed the capabilities of most RT enzymes. Sigma offers a wide variety of kits, reagents, or Master Mixes, each capable in their own way of tackling these difficult transcripts.

Our products incorporate Moloney Murine Leukemia Virus (M-MuLV), Avian Myeloblastosis Virus (AMV), or Enhanced-AMV RT enzymes, each engineered to best create cDNA from difficult RNA. RT enzymes extend DNA primers that have been annealed onto a RNA template, and in this was can create cDNA. M-MuLV is often preferred for longer transcripts (>5kb) because in comparison to AMV it displays reduced RNase H activity. AMV RT enzyme actually has a higher thermostability and as a result is preferred for  transcripts that have difficult secondary structure. Our RT enzymes have been engineered to provide quality first-strand synthesis; the enhanced AMV RT enzyme is stable at higher temperatures and is ideal for low-abundance and difficult secondary structure transcripts. Choose from cDNA synthesis products or RT-PCR products with reagents for all the steps of the process.

Reference

  1. Rapley, Ralph, and David Whitehouse, eds. Molecular biology and biotechnology. Royal Society of Chemistry, 2014.


 RT-PCR Kits

Our RT-PCR kits provide everything needed to perform reverse transcription followed by PCR analysis of the synthesized cDNA. Choose our Enhanced AMV kit for difficult experiments with low abundance transcripts or difficult secondary structures. Or if you’re looking for something more convenient, check out the Titan One-Tube options for convenient, high-fidelity reverse transcription and amplification.
 

Product No. Product Name Features
HSRT100 Enhanced Avian HS RT-PCR Kit
• Flexible kit for one or two step RT-PCR
• Highly purified eAMV-RT - transcribe difficult secondary structure at high temp
11939823001 Titan One-Tube RT-PCR Kit
• Reduces the error rate in PCR due to the proofreading capability
• Threefold higher fidelity than standard Taq
11855476001 Titan One-Tube RT-PCR System
• Reverse Transcriptase AMV for first strand cDNA synthesis
• Expand High Fidelity Enzyme with proofreading for cDNA amplification

 

 cDNA Synthesis Kits/Mixes

Our kits and mixes provide a wide variety of options to meet your reverse transcription needs. Choose from various types of RT enzymes as well as several different packaging options for your convenience. All of these products include everything necessary for first strand synthesis and are available as kits with separate reagents for optimization or as complete mixes for quick and convenient First-Strand synthesis.
 

Product No. Product Name Features
STR1 Enhanced Avian First Strand Synthesis Kit • Highly purified eAMV-RT - transcribe difficult secondary structure at high temp
• Detects low abundance mRNA.
GE27-9261-01 First-strand cDNA Synthesis Kit • Pre-assembled bulk reaction mixes to save time and preven contamination
• Includes two primers for mRNA with or without Poly(A) tails
11483188001 First Strand cDNA Synthesis Kit for RT-PCR • Includes AMV RT for higher thermostability vs. M-MuLV RT
• Includes Rnase Inhibitor and dNTP Mix for PCR
RDRT ReadyScript™ cDNA Synthesis Mix • Sensitive and easy-to-use solution for two-step RT-PCR
• ReadyScript Enzyme is an RNAse H(+) modified M-MuLV RT
GE27-9263-01 Ready-to-go™ T-Primed First-Strand Kit • 50 single-dose, ambient-temperature-stable, first-strand synthesis reactions
• Each contains Notl-(dT)18 Primer, Cloned FPLCpure M-MuLV RT, RNase Inhibitor, and dNTPs

 

 RT Enzymes – Reverse Transcriptases

Choose from our collection of RT enzymes and find one that suits the complexity of your RNA transcript. Our products include standard Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus Reverse Transcriptases as well as enhanced versions of these for higher sensitivity.
 

Product No. Product Name Features
A4464 Enhanced Avian Reverse Transcriptase • Greater sensitivity for low abundance mRNA
• Best enzyme on market for transcribing through difficult secondary structure
ERT-RO Expand™ Reverse Transcriptase • Transcribe cDNA fragments up to 13.5 kb
• Can use cDNA or single-stranded DNA
M1302 M-MLV Reverse Transcriptase • Uses single-stranded RNA, DNA or RNA-DNA hybrid
• Generate first strand cDNA up to 7 kb
10109118001 Reverse Transcriptase AMV • Includes 5X First-strand cDNA Synthesis Buffer
11062603001 Reverse Transcriptase M-MuLV • Lacks endonuclease activity and has much lower RNase H activity vs AMV RT

 

 Additional Reagents

Along with our kits and RT enzymes, Sigma also provides a variety of reagents to meet your experimental needs. RNase inhibitor and primer options are available to further augment your RT-PCR experiment.
 

Product No. Product Name Features
10814270001 Primer for cDNA synthesis • Oligo-p(dt)15
• Binds the 3′-poly-A tail of an mRNA to initiate synthesis of a cDNA
R7647 Random Nonamers (cDNA Primers) • Random sequences of nine deoxyribonucleotide
• May be used as universal, non-specific primers
RNAINH-RO Protector RNase Inhibitor •  Inhibit RNases during RNA isolation and purification
•  Active at pH 5.0 to 9.0 and at temperatures between +25°C to +55°C
R2520 Ribonuclease Inhibitor - Human • Useful for in vitro inhibition of ribonucleases
10786357001 Ribonuclease H (RNase H) • Eliminate potential sources of PCR errors
• Increase accessibility of primers during subsequent PCR