One step and Two step RT-PCR
Real time RT-PCR is the key to success when analysis of the gene expression is the question, which starts with tedious whole cell RNA isolation. Cannot get good RNA yield or having difficulty troubleshootin? Visit here for RNA isolation and purification troubleshooitng guide. Don´t have the time and enthusiasm for RNA isolation? Try our HotScriptase RT polymerase and HotScriptase RT PCR from cell master mix! For its application and protocol, visit here > If you cannot choose a perfect method for RNA extraction , a comparitive take on various RNA extraction methods may help you.
In olden times the RT-PCR could be only performed in 2 steps (reverse transcription carried out in a separate reaction). The first being the reverse transcription and the second being the real time PCR. The reverse transcription was first performed with different buffer in a different tube for reverse transcriptase and then an aliquot of the cDNA produced in the first step was used to further to run the real time PCR with DNA polymerase with a different buffer (two steps). This is quite labour intensive and error introducing procedure along with contaminants that could be introduced at each step due to the transfer of tube content from one to another. Also opening and closing of tubes is a potential source for the introduction of contaminants. However it was more specific and sensitive.
Nowadays we have a faster version where both the steps are combined into one and the whole experiment can now be performed in 1 step (reverse transcription carried out in the same tube as PCR). In this case the time is significantly reduced and the setup is much easier due to reduced pipetting steps. This also accounts for very less chances of contamination, since all the reagents and templates are introduced all at once. However since the buffer is optimised for both the enzymes to work, the method isn´t as sensitive as the 2 step RT-PCR. This is why one must take all the points into consideration before choosing the type of RT-PCR. For Genaxxon´s cDNA one step RT-PCR mastermixes and kit, visit here >
The table below compares the two types of RT-PCR!
|Properties||1 step RT-PCR||2 step RT-PCR|
Less experimental variation due to both reactions taking place in one tube
More experimental variation due to change of tube and several pipetting step.
|Low probability||High probability|
Suitable for high throughput amplification
Less sensitive as the buffer is optimised to function for both the enzymes but the gene-specific priming may be more sensitive for quantification of certain genes.
|More sensitive as the buffer in the two steps are optimised for the two different enzymes|
|Troubleshooting||Almost impossible due to the combination of both the steps||Easy as the two steps are separate with different reagents at each step|
1) Clementi M, Menzo S, Bagnarelli P, Manzin A, Valenza A, Varaldo PE. Quantitative PCR and RT-PCR in virology. PCR Methods Appl. 1993;2(3): 191-196. doi:10.1101/gr.2.3.191
2) Huggett, J., Dheda, K., Bustin, S. et al. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6, 279–284 (2005). doi:10.1038/sj.gene.6364190