One step and Two step RT-PCR
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 |
|
Experimental variation |
Less experimental variation due to both reactions taking place in one tube |
More experimental variation due to change of tube and several pipetting step. |
|
Contamination |
Low probability |
High probability |
|
Speed |
Fast |
Slow |
|
Reproducibility |
High |
Low |
|
Suitable for high throughput amplification |
Yes |
No |
|
Sensitive |
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 |
References:
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
Zugehörige Produkte
