Hi everybody,
Recently I move on to a new projects about circRNA and a disease, very interesting. My boss got a company to sequencing every published circRNA with patient's samples, around 1000 show different expression level compaired with control.
After I got results from the company, I choose 10 most up regulated circRNA and try to confirm them on my cell models, then I got a problem.
I set up cell models and extract total RNA with Qiagen RNA kit, then RT them to DNA with random primers (TAKARA). I didn't use a RNase-R treatment because my boss think divergent primers should not have any problem with remain mRNA.
So I designed divergent primers for each circRNA and run normal PCR for 30 cycles. I pour an agar gel and get mutiple bands. Each primers were blasted with NCBI database and they should be single.
Then I got a advise, one of my friends said I may amplified other circRNA which come from same gene, they may share same exon. So I use circbase data, and found what he said was true, I do get other circRNAs.
So I re-designed new divergent primers, and this time, each pair of them has 1 primer just at joining site. After checked with circbase's data and NCBI's database, I should have a single band now. I also changed agar-gel to 10% DNA-PAGE, because new primers yeld small PCR products (around 80bp).
Here is the problem, I still got mutiple bands....... and they around 50-400bp. I got my target band, and I also get others. Really have no idea what happened. Did anyone has experience for circRNA? Is that because I didn't use RNaseR? Any reply will be help.
Thanks
I don't have any working experience on circRNA. But I think that this may happen for the presence of other RNA types, like long noncoding RNAs. You can check the database and identify any unwanted hit. Also random primers create lots of unwanted cDNAs, you can try specific primer during reverse transcription to make the next step (PCR) more specific.
Please check your RNA quality and be sure it has no contamination with other RNA rather than your target one. Best of luck
Hi Monica, I did not catch your question earlier and is afraid that you have already solved your problem :).
I think that that disregarding specific or random RT primers used, the reverse transcription of circular RNA templates could result in synthesis of multimeric cDNA templates (via RCA mechanism). PCR amplification of multimeric sequences with divergent PCR primers will always produce shorter and longer PCR amplicons because of multiple binding sites for the same primers on such templates containing repeats of the circular RNA (antisense) sequences. Possible ways to predominantly get only the shorter PCR amplicons are using the shorter primer extension time (in PCR cycles) and/or 5’-exo minus Taq Polymerases.
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