to perform RT PCR to RNA sample the used technique 1st convert RNA to DNA in separate reaction then perform replication
can i make them in one step?
The disadvantages are huge as mentioned in the above link:
- Must “start over,” or save RNA aliquot and perform new RT to analyze new target or repeat amplifications
- Reaction conditions are not optimal—efficiency & thus quantification are affected
- Primer dimers a bigger potential problem
Remember, if you haven't characterized your primers and there is potential issues with it, you will have to perform the RT reaction again. You should also check whether the kit is compatible with one-step because in some kits it may suggest you to dilute the cDNA to lower inhibitory effects of salts used in the cDNA synthesis process.
It is possible , you can use kit contains all component necessary for cDNA synthesis and amplification in one tube such as ACCuPower RT/PCR kit
I agree, it can take time to optimize, however if you are looking at multiple samples, where volumes are limited, or very dilute (for example with RNA viruses), the one-step is the recommended method.
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