I am extracting viral RNA from a human tissue. I have DNA elute. Now if I give DNASe 1 treatment, how do I proceed with further elution of DNA free RNA?
Hello Heena,
RNA will be extracted together with DNA. Personally, after DNAse inactivation (75°C for 10'), as a first test I would **try** to go straight to RT, possibly by using Oligo(dT), to get rid of 'contaminating' human ribosomal RNA, assuming that your virus has a poly-A tail as is often the case with RNA viruses.
Hello Heena,
also I agree wih previous answer.
http://www.ncbi.nlm.nih.gov/pubmed/21404095
this was our previous work and in this case we did twice RNA Digest after RNA extraction. but as you know, we have used bacterial RNA not human sample.
Hello Heena
i also agree with Rocco. Remember though to heat inactivate your DNAse at 85c for 10 min before commencing your RT reaction with Oligo dT
My concern would not be so much specificity as efficiency of RT synthesis. I would spec/ nano drop my DNA column eluted DNAse digested sample and add 2ug to an Oligo dT RT reaction: if this was just 2-5ul in a 20ul reaction then buffering conditions would be compatible with efficient RT. Alternatively and better still try doubling the amount of buffer and making your total reaction volume 40ul to minimise the impact of impurities on your RT reaction
If this doesn't work try selectively precipitating your RNA with LiCl and then subjecting to 70% ethanol wash ( to remove the salt) before re suspending the much purer sample in water. Then Nano drop and then for reasons given by Rocco carry out RT using Oligo dT and 1ug of your precipitated sampke
see link below for details of selective RNA ppt using LiCl
Ppt protocol and explanation:
https://www.thermofisher.com/uk/en/home/references/ambion-tech-support/rna-isolation/general-articles/the-use-of-licl-precipitation-for-rna-purification.html
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