My RNAseq data and qPCR showed that there is an up-regulation of one lincRNA in the treated cells but when I want to amplify the full length of this lincRNA (1600bp) by RT-PCR, I have no band. Do you have any suggestion?
any tips for cDNA synthesis or a specific DNA polymeras?
Is lincRNa amplification different comparison with mRNA amplification?
Not all lncRNA have polyA tails, so if you are not seeing a product using oligo-dT primers you should try using random hexamers instead during cDNA synthesis. If that still doesn't give you a band, you'll need to check the design of your cloning primers.
I would recommend using a high-fidelity DNA polymerase (such as Q5) for all cloning PCRs.
I agree with John's advice. Also wanted to add that if the purpose of the experiment is cloning the full-length RNA, you can use a gene-specific primer for reverse transcription that is designed against the 3' terminus of the target RNA. This is easy and cheap, and increases your likelihood of getting a full length cDNA for a transcript that is not polyadenylated. Also, since some lncRNAs are highly structured, you could try increasing the reaction temperature of the reverse transcription and make sure you are using a reverse transcriptase that copes with high temperature and secondary structure. SuperScript III at 50C with a gene specific primer nearly always works for us. Also, check that your input RNA is of high quality, because getting a full length cDNA from degraded template is difficult and maybe impossible. Good luck!
Thank you all for your suggestions! I optimized PCR with changing of Enzyme and primers and it worked finally ! :)
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