I'm relatively new to Reverse transcription. I have worked with cDNA before but dealing with RNA extraction and reverse transcription is new for me so bare that in mind.
I'm studying the expressión of a gene in some mouse tissue. At the same time, I'm getting samples from the cochlea and the vestibular apparatus, which is known to express a significant amount of this gene, as a means for positive control. I carry RNA extraction with Tranzsol (some kind of Trizol rip-off) and it goes well, RNA integrity looks fine and I'm getting about 200-5000 ng/ul total RNA depending on the tissue I'm working with (measured with a picodrop). A260/280 is about 1.7-1.8. The spectra looks good without any significant amount of guanidinum salt carried over.
I then use 1ug of total RNA for RT. My protocol goes like this: 1. gDNA digestion with DNAseI incubated with some of the RT buffer (37ºC, 30 min). Following deactivation at 75ºC for 15 minutes. I then add some more buffer, water, dNTPs and OligodT and do 5 minutes at 65ºC. Now I get to add the RT enzyme (Easyscript from Trans), I do a negative control without the enzyme and finish off with RNAse H.
When I get to test this cDNA (by end point PCR), I always get a nice short bp band for GAPDH which doesn't show up on the negative control (-RT), which is good news because that means that I'm not having genomic DNA. But when I'm testing my gene of interest on the sample I got from the control tissue I don't get the same band as the cDNA from the same control tissue that my PI brought from germany. He used better kits (Superscript), but my cDNA seems to work just fine with GAPDH primers. NTC controls for both sets of primers are clean and I'm mastermixing everything.
Some people in the lab suggested that there could be unsufficient time for the retrotranscription of the mRNA I'm looking for if my primers are too close to the 5' end (which they might be, I think I got them from the first 2 exons), while GAPDH mRNA get's fully converted to cDNA just good.
What do you guys think? I apologize in advance for my poor english.
Try using a cDNA kit that uses random primers instead of OligoDT primers.
Time has never been an issue in terms of reverse of transcription.
Hi Esteban,
Rather than insufficient time for RT, it could be that the template the RT is working on is less than ideal. Picodrop tells you the amount of nucleotides and gives a good indication for contamination - but it doesn't actually tell you if your extracted RNA is intact. If your samples are degraded, you can have the best downstream procedures in place but you will still only get fragmentary or no amplification when you hit the PCR stage. I would highly recommend sending off your samples to bioanalyzer before going onto RT to see if the mRNA is intact.
This kind of thing has happened to me a number of times - the samples look excellent on the nanodrop, with incredibly high RNA concentrations and no contamination. But when I sent them to the bioanalyzer the samples were degraded and completely inadequate for further experiments. If you send yours off and find that the samples aren't degraded, that also gives you a better idea where you can go on from there for troubleshooting.
Good luck!
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