I ran a PCR of reverse transcribed RNA samples using a primer combination that should amplify two splice variants of a gene.
One of the splice variants should be 399bp in size, while the other should be 220bp.
But when I ran the gel, I found a third band had also been amplified, around the 100bp region (see image attached).
I do not think it is primer dimers because I used a dH2O control (mastermix+primers+water instead of sample) to detect contamination in the mastermix and primer dimers, and there is no band on this lane.
Does anyone know what this band could be?
Or at least, does anyone know if this could be a genuine mRNA and not just an artifact?
They appear to be tRNAs judging from the size/smear. Is it possible that there is tRNA present in your samples?
Dear Miguel Hernandez Madrigal
Providing some more details such as: what type of PCR did you run? Were the primers designed within exons or spanning the splicing region? etc, could help us to understand what is going on in your PCR, and thus discard possible causes.
Judging the picture, I would suggest you to check the specificity of both primers, since there is not only two type of bands. Primers not only hybridize to form dimers, they can also self annealing to form hairpins that can be amplify as artifacts, 100 bp apply for such a case. In a second approach, clone each band produce in the PCR run, and send then for sequencing, then make a blast to discover what they are related to.
Hope I have been of help.
best.
Thanks for all the replies.
The sample material for the PCR was RNA extracted from a cell lineage using column extraccrion method. They were then reverse transcribed into cDNA using random hexamers.
The primer pair is designed to span across exons. Forward primer is located in Exon 1, reverse primer is located in Exon 3. Exon 2 can be spliced out, which leads to an amplicon of expexted 399bp (from transcripts containing exon2) and another one of 220bp (from transcripts without Exon 2). I did not expect any more amplicon.
I don't think the band at 100bp is primer hairpins or dimers or other structures because there are no bands of this size in the control.
I still could be an artifact, but i don't know which type of artifact.
Hi Miguel Hernandez Madrigal
Thank you for the details added. You are right, definitely not any artifact derived from primers.
I am wondering, whether did you apply any DNAse treatment? and, if you blast your primers how many hits do you get?
Good weekend
Thanks again Leonardo Martin Martin
I did not apply DNAse treatment.
With regards to the primers, I used to get two hits only (399bp and 220bp fragments), but recently ncbi updated the transcript of this gene on their database, shortening exon 1 and leavingmy forward primer "out" of the accepted exon1. Because of this, now when I blast my primers I get 0 hits.
I do get amplification from these RNA samples nonetheless, and expression levels change between on qPCR, which leads me to believe that the "old" accepted sequence is still correct, but for some reason they decided to shorten it.
In summary, no DNAse treatment, and only 2 hits.
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