Many cellular functions are regulated by gene expression. Diseases as auto immune disorders, chronic inflammatory diseases or viral infections (HIV, HSV) alter most often the transcription of affected cells. Therefore quantitation of the mRNA level of a specific gene or of viral mRNA has become an important and common method in clinical diagnostics respective in modern molecular medicine.
Reverse transcription is the synthesis of single-stranded DNA (complementary DNA, or cDNA) using RNA as a template, mediated by reverse transcriptases (RTs) or the newly developed HotScriptase RT from Genaxxon. The cDNA can be used as a template for amplification by PCR or to generate a cDNA library.
Using realtime PCR with fluorescence marker will allow the quantitation of the original RNA content of a sample.
With the newly developed HotScriptase RT from Genaxxon bioscience reverse transcription and subsequent PCR will become much easier and additionally will allow to quantify even RNA that shows very stable secondary structures that are stable above 40°C. Using HotScriptase RT RNA will be transcribed into DNA and amplified in one single procedure using a simple standard PCR protocol. HotScriptase RT allows therefore a “Zero-Step” RT-PCR without the necessity of an isothermal reverse transcription step. Using appropriate Probes or Primer quantitation of the original RNA content can be accomplished directly. No additional pipetting or programming is necessary to get quantitative data.
RNA is always a very precious product that is difficult to gain and hardly stable which makes it desirable to have the possibility to overcome tedious RNA preparations. With the HotScriptase RT Cell Mastermix Genaxxon offers a tool that enables to overcome this problem. With the HotScriptase RT Cell Mastermix it is possible to execute RT-PCR without isolating RNA before reverse transcription, respective before quantitation of RNA by qPCR, just by using cells. For this cells will just be mixed with our HotScriptase RT Cell Mastermix and the mixture placed into a realtime PCR instrument for subsequent PCR.
HotScriptase RT Polymerase and HotScriptase RT Cell Mastermix will substantially simplify and easing the established but old method of RT-PCR and in addition gives more flexibility, reliability and possibilities regarding quantitative PCR.
The term RT-PCR is sometimes also used for RealTime PCR which may cause some confusion as this term has been used before for the combined reverse transcription with subsequent PCR. For that it is better to use qRT-PCR for the quantitative reverse transcription with subsequent realtime PCR.
Find out more about our products for qPCR.