I was trying to synthesize cDNA from an RNA extracted from Arabidopsis thaliana.
The RNA was digested with Ambion Turbo DNase and extracted with chloroform.
The digested RNA was used as template in PCR and the primers can give bands with different size depending on the template is genomic DNA or cDNA. The result was that there was no genomic DNA band (in fact, no bands at all.)
However, when I synthesized cDNA with this digested RNA, the resulting cDNA can give both cDNA band (smaller) and genomic DNA band (larger) in PCR reactions.
The picture showed the double bands in cDNA samples, and a positive control with genomic DNA. In the negative control there is no band.
Could anyone tell me what is the reason?
Oh, no you definitely want them to span introns. If they don't, you won't be able to distinguish genomic from transcript at all.
You just want them to span LARGE introns. I usually try to find introns that are 5000bp or more, ideally larger. So: "forward primer that binds to exon1, 150000bp of intron, reverse primer that binds to exon2" for instance. There'd be no risk of detecting unspliced or genomic template, because a 150000bp PCR product is never going to form.
Another approach is to design primers that only recognise exon:exon junctions (so bind to a bit of sequence at the end of exon1, and a bit at the start of exon2): these are trickier to design, because your options are more limited, but these will only detect spliced transcripts.
Assuming you're distinguishing cDNA from genomic DNA via intron-spanning primers, this is the sort of thing you'd see if the intron in question is very small. Since splicing doesn't necessarily immediately follow transcripton, you will often have some RNA molecules that still contain introns. Turn these into cDNA, and if the intron is small enough, these will still represent viable PCR templates.
Of course, if you're only analysing using gel electrophoresis (which is frequently run under conditions sufficient to detect single transcripts), you don't necessarily know how MUCH of this template you have. It might only be a few molecules (vs several thousand of the spliced transcript).
I don't think that the upper bands in your cDNA samples originate from genomic DNA. If this would be the case the upper band would be of a much higher intensity.
My opinion: these upper bands could perfectly be dimeric configurations of your product. In the last step of the PCR program cross-hybridizations might occur.
Regards, Christian
Oh, no you definitely want them to span introns. If they don't, you won't be able to distinguish genomic from transcript at all.
You just want them to span LARGE introns. I usually try to find introns that are 5000bp or more, ideally larger. So: "forward primer that binds to exon1, 150000bp of intron, reverse primer that binds to exon2" for instance. There'd be no risk of detecting unspliced or genomic template, because a 150000bp PCR product is never going to form.
Another approach is to design primers that only recognise exon:exon junctions (so bind to a bit of sequence at the end of exon1, and a bit at the start of exon2): these are trickier to design, because your options are more limited, but these will only detect spliced transcripts.
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