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Reverse Transcription and RT PCR

The basic difference between PCR and RT-PCR is the template: DNA is the PCR template and RNA is the RT-PCR template. The first step in RT-PCR is the synthesis of single-stranded antisense DNA from an RNA template. This process employs reverse transcriptases (RNA-dependent DNA polymerases). The Moloney-Murine Leukemia Virus (M-MuLV) reverse transcriptase is a classic example of such an enzyme.

Mutations have been introduced into reverse transcriptases genes to increase performance. biotechrabbit offers a modified reverse transcriptase, RevertUP™ II Reverse Transcriptase, which is a proprietary modification of the M-MuLV reverse transcriptase with point mutations in polymerase and RNAse H domains. These mutations ensure elevated cDNA synthesis efficiency and eliminate RNase H activity, allowing successful synthesis of ≥14 kb cDNA.

cDNA can be synthesized by reverse transcriptase either using random primers, oligo-dT primers or sequence-specific primers.

Special care should be taken to prevent RNA templates from degradation by pervasive RNases. Therefore it is always a safe approach to use RNase inhibitors in all reactions involving RNA.

RT-PCR can be performed in one step or two steps. Two-step RT-PCR includes two different experiments, first the reverse transcription of RNA template is performed and then the PCR amplification of the cDNA template is performed in another tube. One Step RT-PCR combines both reactions in one tube: cDNA is synthesized from RNA and then amplified to generate cDNA with specific primers without additional pipetting steps.

Taq DNA Polymerase and its hot-start versions are the main enzymes used for cDNA amplification in both the one-step and two-step RT-PCR.

More information:
Reverse Transcription and RT PCR Product Selection Guide
Product Application Main Features
One Step RT PCR Kit One step RT PCR A blend of efficient thermostable Reverse Transcriptase and proprietary RNase Inhibitor provides high cDNA yields. Unique Hot Start Taq DNA Polymerase in a mix with high quality dNTPs and PCR enhancers allows sensitive low background amplification.
RevertUP™ II Reverse Transcriptase, 200 u/µl Reliable synthesis of ≥14 kb cDNA, two-step RT-PCR A proprietary M-MuLV reverse transcriptase engineered by point mutations in polymerase and RNAse H domains that ensure efficient cDNA synthesis with no RNAse H activity, allowing successful synthesis of ≥14 kb cDNA
MMuLV Reverse Transcriptase, 200 u/µl cDNA synthesisTwo step RT PCR A classical exceptionally pure Reverse Transcriptase supplied with the Reaction Buffer
RNase Inhibitor, 40 u/µl Prevention of RNA from degradation by RNases A recombinant non-competitive inhibitor of pancreatic-type ribonucleases such as RNase A, RNase B, and RNase C
Taq DNA Polymerase, recombinant, 5 u/µl Amplification of cDNA Exceptionally pure recombinant Taq DNA Polymerase, supplied with separate Magnesium to allow flexibility in reaction optimization
Hot Start Taq DNA Polymerase, 5 u/µl Hot Start amplification of cDNA Exceptionally pure antibody-based Hot Start Taq DNA Polymerase, supplied with separate Magnesium to allow flexibility in reaction optimization and with the PCR Booster for even less background