RT-PCR — Reverse transcription PCR

What Is RT-PCR?

Reverse transcription polymerase chain reaction (RT-PCR) is a variation of standard PCR that involves the amplification of specific mRNA obtained from 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 enables the production of cDNA from the RNA sample by reverse transcription. This cDNA anneals to one of the primers leading to first-strand synthesis. Standard PCR proceeds and dsDNA is produced.

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 virology diagnostic method and is frequently combined with quantitative real-time PCR (qPCR), which is widely used to quantify RNA transcript levels in cells and tissues.1 The combination of real-time PCR (qPCR) and reverse transcription PCR is known as quantitative RT-PCR or qRT-PCR.

 

Working with Difficult RNA Transcripts

Low abundance or complex secondary structure of RNA transcripts may exceed the capabilities of reverse transcription enzymes. The 3 kb genome region from the RNA polymerase gene to the 3' poly(A) tail of small round-structured virus (SRSV) can be difficult to reverse transcribe because of a stable secondary structure in a region between the RNA polymerase gene and the 5' end of the second open reading frame. We offer a wide variety of kits, reagents, and master mixes capable of addressing RNA regions that are difficult to transcribe.

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

RT-PCR Kits

All the reagents needed to perform reverse transcription followed by PCR analysis of the synthesized cDNA are included in our RT-PCR kits. Choose the Enhanced AMV kit for difficult experiments with transcripts in low abundance or with complex secondary structures. The Titan One-Tube options offer 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 temperature
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
TOSRTRO Transcriptor One-Step RT-PCR Kit • Uses hot-start one-step RT-PCR technology to ensure sensitive and robust reverse transcription.
• Provides all the components required for one-step RT-PCR.
KK4752 KAPA PROBE FAST One-Step • Designed for high-throughput, fast-cycling, one-step RNA quantification.
• Suitable for use with all fluorogenic probe-based technologies, including hybridization probes (e.g., FRET), hydrolysis probes (e.g., TaqMan®) and displacement probes (e.g., molecular beacons).

 

cDNA Synthesis Kits & Mixes

To meet your reverse transcription needs, choose from various types of enzymes and several packaging options. All of these products include the reagents 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 temperature
• 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
• Contains Notl-(dT)18 Primer, Cloned FPLCpure M-MuLV RT, RNase Inhibitor, and dNTPs
11117831001 cDNA Synthesis Kit • Optimized one-tube procedure for the synthesis of double-stranded cDNA up to 3 kb from total RNA or mRNA.
THIFICDNARO Transcriptor High Fidelity cDNA Synthesis Kit • Designed to reverse transcribe RNA with increased fidelity compared to other reverse transcriptases.
• Optimized for two-step RT-PCR
05893151001 Transcriptor Universal cDNA Master • Convenient fast solution for cDNA synthesis and real-time PCR analysis. Whichever PCR instrument you are using, this master mix, supplied in two vials, realizes the full potential of your qRT-PCR assays.

 

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 and 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 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
TRANSRTRO Transcriptor Reverse Trascriptase • Achieve high sensitivity in two-step RT-PCR. Transcriptor Reverse Transcriptase is used in conventional thermal cyclers and real-time PCR instruments (e.g., the LightCycler® Instruments).
• Reverse transcribe difficult templates. The enzyme works well at elevated temperatures, thereby overcoming RNA secondary structure (e.g., GC-rich RNA templates) and facilitating optimal reaction conditions.

 

Additional PCR Reagents

Along with our kits and RT enzymes, we also provide a variety of reagents to meet your experimental needs,including RNase inhibitor and primer options.
 

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 5-9 and at temperatures from 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
W4502 Nuclease-Free Water • Molecular grade
• DNase-free, RNase-free, Protease-free

 

Reference

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