Hi, I want to ask if degraded RNA is able to synthesize cDNA by RT PCR? If yes, so when we use this cDNA and do PCR for cloning, so it affect original pcr product size etc? I want to clone one gene whose size is about 480 bp but when i use cDNA and after running gel it give me size of about 500 bp. RNA is not degraded, don't know where is mistake? Please help me, its urgent
Hi Bushra,
1. If the cDNA of your gene-of-interest is not synthesized full length, you got a 'partial' of your product, and it won't consider as a 'correct' product.
2. As said by other people, it is very difficult to differentiate 500-bp from 480-bp by gel information. Even your product is line up with a 500-bp DNA marker band, it could be just a 480-bp DNA itself in reality. Sequencing can tell you the exact bp numbers.
3. If the 20-bp difference is real, you might also have an 'alternative splicing' variant (see attachment). For example, a 20-bp intron did not splice away in this variant. You should sequence it to find out. After you clone it, you have to sequence it anyway to confirm the right DNA sequence before use it for downstream experiment.
Rverse transcriptase syntheses the cDNA from the 3' end of mRNA, so if the RNA degradation occurred at the 5'end, it wouldn't affect the cDNA synthesis but you will get the truncated version (short, not full length). In most case cDNA is generated using polydT primer and usually contains some promoter sequence upstream of the gene translation start codon (ATG). In your case it is more likely that you got the right gene product since a 20-basepair variation of 500 bp DNA fragment could be resulted by sample buffer difference during your electrophoresis.
It depends on how degraded it is, and whether your transcript of interest happens to still be there or not. If you do have degradation I would not risk it. I strongly suggest checking your RNA on a bioanalyzer.
When you are saying the whole size of the product is expected to be about 480 bp, does that include the ~40 bp of the primer annealing sites from PCR? A common mistake is that folks assume that only the area between the primers will be part of the amplicon.
Also, remember if you ligating a sticky end (rather than a blunt end) into a plasmid there will still be some bases left over after a digest.
Actually i am working on wheat have A B & D genome, so is it possible that product size variation is due to different genome?
I strongly recommend running RNA con a bioanalyzer to check its integrity.
If the RNA is degraded, no matter which priming strategy you use for RT, you will get shorter cDNA fragments, and these might not contain full length products for your your fragment of interest (say, cDNAs that include both primer annealing sites for the PCR).
Nevertheless, a difference between 480 and 500 bp is difficult to tell when running agarose gels. The 480 bp fragment you see might actually be your 500 bp fragment of interest. You can check that easily through sanger sequencing prior to cloning.
@Iker Garcia it means if cdna is not full length it can not tell us about our correct product? What about in my case i get bands same as 500 bp so is there something wrong?
Hi Bushra,
1. If the cDNA of your gene-of-interest is not synthesized full length, you got a 'partial' of your product, and it won't consider as a 'correct' product.
2. As said by other people, it is very difficult to differentiate 500-bp from 480-bp by gel information. Even your product is line up with a 500-bp DNA marker band, it could be just a 480-bp DNA itself in reality. Sequencing can tell you the exact bp numbers.
3. If the 20-bp difference is real, you might also have an 'alternative splicing' variant (see attachment). For example, a 20-bp intron did not splice away in this variant. You should sequence it to find out. After you clone it, you have to sequence it anyway to confirm the right DNA sequence before use it for downstream experiment.
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