Hi,
I am making a DNA modification using DNA ligations, then making invitro produced RNA, capping it and transfecting the RNA into cells. Then I do a total RNA extraction followed by reverse transcription. The reverse transcription of the RT product against the unextended primer is shown in the figure attached here.
I seperate and gel purify this RT product and circularize it and do a PCR. I see that my control vs RT PCR shows empty libraries in both. I dont know how this can be possible.
Am I missing an obvious detail?
Hi,
Re-read throughly the protocol you are following. See if you have someone close that you can talk with for additional doubts. Check the integrity of RNA (I think that it should be best for you to purify mRNA because in your gel it seems to have to much rRNA). And, finally, check if you can possibly be having problems with PCR inhibitors. It will be a good idea also to check a protocol that avoid PCR in order to avoid your bias. Good luck!
Hi,
Thanks for the help. This is my reverse transcription gel. I have checked my RNA quality before and it looks good. Here I am trying to extend a particular kind of RNA which does not include rRNA .
Do you just re-circularize the RT product or use some vector? What is the end result that you expect from this procedure?
I circularize the RT product , there is no re-circularization. The end product is a library for next gen sequencing
Ok, you could try using a vector to make like a cDNA library. It may be a little lenghty process but atleast would ensure that your RT product is maintained which you could then sequence.
Hi,
I use the superscript 3 RT . I can't use a kit as I am interested in making libraries respective to a particular transcript set.
Thanks for your help.
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