i extract RNA from animals then used Bioline cDNA synthesis kit,,, i want to make sure about cDNA synthesis... i measure DNA using nanodrop gives reasonable range but make gel electrophoresis gives bands with some samples and smears with others... what is this mean? and what should i do?
Run one PCR using house keeping gene primers like beta-action or GAPDH.
One more time checking genomic DNA contamination status of regular PCR with reference genes. Most of kit or general steps do not completely removed genomic DNA.
You can treat produced cDNA solution with RNаse (about 30 min) and then do the agarose gel electrophoresis. If you see the clear band of the exact length - most likely you have specific cDNA. Smears mean degraded RNA or DNA - you obtain very low amount of cDNA or no cDNA.
May be you can purify your RNA before cDNA synthesis. Also use a bioanalyzer to check the RNA integrity if possible. The quality of your cDNA depends on how good your RNA is. I tend to go with the A280/260 and A260/230 for he quality of my RNA and cDNA when I nanodrop. Did you mean these values were between the range you expected?
other option also you can use live vaccine as positive control to confirme the cDNA kit the action is good
According to me the best option is to check PCR amplification of house keeping gene.
I agreed with Chang-Soo Seong concern that DNA removal kit do not completely removed genomic DNA but if you run PCR amplication at low cycle like 25 cycle there will be no visible amplification from genomic DNA contamination. So you can easily confirm cDNA synthesis.
Another way is if your primer is exon spanning the amplicon from genomic DNA and cDNA will be different.
Good luck.
Hi Nada,
Your original question to me seems to be asking how to measure cDNA concentration in solution.
Oligogreen on a Qubit machine can help you measure single stranded-DNA species (such as cDNA) by fluorometric means.
qPCR can indeed also indicate extent of cDNA synthesis as others have mentioned above.
If random priming + oligo dT was used during RT, a striated smear would be expected (along with 2 distinct ribosmal bands of appropriate size); the striations are merely the other sized cDNAs made from the myriad of other sized mRNAs. Dinstinct bands without smearing seems to indicate you used a gene-specific primer during RT. Without knowing how you prime your RT rxns, this is all I can comment on. Measuring cDNA by spec becomes nonsense as dNTPS, left over RNA and NTPs, and cDNA all contribute to the A260nm reading...
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