For RT-PCR the RNA molecules are transformed into their complementary DNA (cDNA) sequences by reverse transcriptase. (with Mulv)
The next step is the amplification of the newly synthesized cDNA by standard TAQ polymerase PCR procedures.
This approach to examine gene expression is universally known as RT-PCR, because of the role of reverse transcriptase (RT) in the synthesis of first-strand cDNA.
What is the procedure for RT-PCR?
The ranscription of purified RNA by RT reverse transcriptase via an appropriate method for priming and amplification of cDNA with some form of PCR.
What is Normalisation in RT-PCR?
Normalization of samples is essential in RT-PCR, and the efficiency of first-strand cDNA synthesis is one of the most crucial determinants in the success or failure of this method. The initial quantity of RNA in the sample needs to be determined.
Because of this, it is to earn a large cDNA pool where aliquots may be drawn for programs rather than repeating the cDNA synthesis reaction repeatedly.
How to design a useful RT-PCR?
The trick to RT-PCR resides in the design of useful primers that market a balance between template specificity, thermodynamic equilibrium when base-paired to the template, and ability of one primer to function with the other(s) to encourage RT-PCR. Given the sensitivity of PCR, it is now commonplace to detect and quantify transcripts present in abundance. Permutations of RT-PCR have been in use.
How to design RT-PCR primers for COVID 19?
The instrument needed for Coronavirus reverse transcriptase rt-Pcr detection of an Rna virus is a thermocycler. Rna is transcribed with reverse transcriotase to cDna copy dna and than analysed.
Thermocyclers are expensive instruments in therms of investment, use and need qualified researchers to work with.
Not all hospitals have a thermocycler available in China.
The primers for Covid-2019 like the ones Genprice Inc. sells are easily made or selected themselves by US researchers.
In 2020 sold only 3 to less developped countries like Mexico with less confident lab biologists. For them it is easier to use Genprices fully optimized protocol.
In the Usa researchers like to design themselves their coronavirus primers and are confident enough to design the Covid 2019 pcr test by themselves. They can buy inactivated positive control coronavirus Rna called Nattrol or nucleic acid test control from Zeptometrix Inc. in Bullalo, NY. who are working in a Covid 2019 Nattrol.
Today only Mers and Sars coronavirus inactivated non infectiouse controls are available.
In Europe the situation is similar. In Germany lab will use their own designed primers and protocols while in Italy or Spain lab biologists will buy Genprice optimised primer sets.
Lieven Gevaert, Mr. Biotechnology