I,m using Applied biosystem Kit for cDNA preparation from plant samples. But unable to got any result ? One my colleague using same Kit for cDNA preparation but from animal samples. Is random primer works for only prokaryotic cDNA preparation, if yes, then how it works for animal cells, but not from plant cells. Please help me for the same problem.
Your question is a little confusing as plants and animals are both eukaryotes. Prokaryotes and eukaryotes both have mRNA with poly A tails, although prokaryotic poly A tails are shorter and not contained on all transcripts of the same variety (2 to 60%). A mixture of random primers and oligo dT works in all situations. Oligo dT priming works in both prokaryotes and eukaryotes, but gives one 3' bias (thus primers for PCR/qPCR are best designed near the 3' end of target transcripts when using only oligo dT priming). As well, random priming alone works for all eukaryotes and prokaryotes, but oligo dT only works on mRNAs containing poly A tails (oligo dT does not work for ribosomal RNA and histone mRNA priming since they do not have poly A tails; only mis-priming events cause some rRNA targets to be transcribed into cDNA during RT). Other options for priming are a mixture of oligo dT and random nonamers or random pentadecamers as well.
What you are likely experiencing is inhibition of your particular reverse transcriptase enzyme by carry-over inhibitors in your plant RNA extracts. If you are using AB's MultiscribeTM RT enzyme, it has a very poor track record in processivity and is very prone to inhibition. Your plant RNA extracts may contain various co-extracted tannins/polyphenols and/or polysaccharides that are shutting down your RT in my opinion... inhibitors that are not present in your colleague's animal RNA extracts. Method of RNA isolation and purification may also have to be further optimized differently with your plant samples.
Your question is a little confusing as plants and animals are both eukaryotes. Prokaryotes and eukaryotes both have mRNA with poly A tails, although prokaryotic poly A tails are shorter and not contained on all transcripts of the same variety (2 to 60%). A mixture of random primers and oligo dT works in all situations. Oligo dT priming works in both prokaryotes and eukaryotes, but gives one 3' bias (thus primers for PCR/qPCR are best designed near the 3' end of target transcripts when using only oligo dT priming). As well, random priming alone works for all eukaryotes and prokaryotes, but oligo dT only works on mRNAs containing poly A tails (oligo dT does not work for ribosomal RNA and histone mRNA priming since they do not have poly A tails; only mis-priming events cause some rRNA targets to be transcribed into cDNA during RT). Other options for priming are a mixture of oligo dT and random nonamers or random pentadecamers as well.
What you are likely experiencing is inhibition of your particular reverse transcriptase enzyme by carry-over inhibitors in your plant RNA extracts. If you are using AB's MultiscribeTM RT enzyme, it has a very poor track record in processivity and is very prone to inhibition. Your plant RNA extracts may contain various co-extracted tannins/polyphenols and/or polysaccharides that are shutting down your RT in my opinion... inhibitors that are not present in your colleague's animal RNA extracts. Method of RNA isolation and purification may also have to be further optimized differently with your plant samples.
Have you checked RNA integrity on gel or any bionanalyzer...? How do you extract RNA, from which plant species and which tissue? I have never ever had a single problem by both using Trizol (or similar products) or Extraction kits like the one from Sigma for example, also when extracting from 'difficult' tissues. I've used Oligo dT only, random primers only and a mix of the two and always worked... again...you should check your RNA first...
Couldn't agree more with Jack. Last time I screened commercially available kits (admittedly a few years ago) I found ABI's High Capacity cDNA Reverse Transcriptase Kit (Prod # 4368813) to be the best for routine RT reactions. The magic of the kit seems to in fact be in the random primer set. I've pulled out and use those random primers for every RT reaction I do regardless of the RT enzyme. I've done standard curves using in vitro transcribed RNAs and observe near 100% perfect RT from either synthesized or cellular RNA for the above cDNA kit. For particularly difficult RT reactions, I haven't been able to beat using Superscript III with the random primers from the above kit (although that RT is expensive). If that high capacity kit isn't working there must be something wrong with the RNA or your PCR primers. Presumably you are using an endogenous control primer set? Because optimizing custom designed primers can sometimes be troublesome, my lab has resorted to just always using a commercially available endogenous control primers as a reliable positive control. We like ABI's taqman GAPDH primers. Never fails for us.
- Badges
- Science method