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Hello,Why do we need to extract the RNA again after RNase R...
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Summer S Mar 14, 2019
Hello,
Why do we need to extract the RNA again after RNase R treatment? Would RTPCR not work if we skip RNA extraction after RNase R treatment? Thank you for your expert opinions.
Why do we need to extract the RNA again after RNase R treatment? Would RTPCR not work if we skip RNA extraction after RNase R treatment? Thank you for your expert opinions.
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Thank you Amaresh for your detailed response, much appreciated.
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0 comments ShareMyriam Gorospe Author Answered Mar 14, 2019
Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, USA
Usually RNase treatment starts with 2-5 ug which is a lot for RT in the control reaction. Isolating RNA from RNase R reaction will give pure RNAs for cDNA synthesis. RNA isolation after RNase R treatment allows you to setup RT reaction with low RNA amount and save the treated RNA for further use.
If you are using 1 - 2 ug RNA for RNase R treatment, you may directly go for cDNA synthesis. I have also tried using the RNase R reaction for cDNA synthesis without isolating RNA and it works well without any significant inhibitory effect on RT-PCR.
Hope this helps,
Amaresh
If you are using 1 - 2 ug RNA for RNase R treatment, you may directly go for cDNA synthesis. I have also tried using the RNase R reaction for cDNA synthesis without isolating RNA and it works well without any significant inhibitory effect on RT-PCR.
Hope this helps,
Amaresh
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