I am working in a project that l use both one step and two steps methods, but l would like to know which of them more useful and specific.
Hello
When we are dealing with one step RT PCR , it means that we are making both RT reaction and the PCR in a one tube mix (Therefore you can notice the First temperature is not a 95C !!!) , this will , to some extent , keep you away from the risk of contamination with faster results , although you need to include target primers with RT mix.
In 2 step RT PCR , you are going to synthesize cDNA only which will be ready for further downstream applications , thus the risk of contamination will be higher but you will get a plenty of options to deal with (e.g. many primer sets )
I hope this could help
A helpful website: http://www.lifetechnologies.com/uk/en/home/life-science/pcr/reverse-transcription/rt-pcr/one-two-step-rt-pcr.html
Hi Noha,
apart from the suggestions above..
1) one step is better than 2 step for RT if you want to take care of contaminations....and it is useful when you are analysing few genes.....
2) However it offers less flexibility when it come to select the types of RT enzymes you want to use eg...if high annealing is desired for compex RNA molecules etc....you don't get both 1 and 2 step kits for all RT enzmyes
3) If you are planning analysis of laarge number of transcripts in a sample....itwould be better to do RT first and then PCR separately for different genes....many factors will be same including cDNA synthesis efficiency ...unlike doing 1step analysis for all the genes..
4) One step analysis for large number of genes will be more expensive than 2 step analysis in the same sample
5) and also ...modt commonly used 1 step RT-PCR kits have mixes with low sp. activity RT enzymes .....if you desire to have high specific activity ot RT...it may not be possible...
so decide the types of analysis and selct the methods......contamination can be taken care by a careful planning of experiments and good lab practices
bye
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