first strand cDNA synthesis not working - cannot PCR out cDNAs of interest from first strand cDNA (Feb/18/2010 )
I need your help in
troubleshooting a cDNA prep I made. I drug-induced my C.elegans worms and then
prepared RNA from them using Trizol, but column-purified using the Qiagen Rneasy kit. I measured RNA concentration by Nanodrop and got
1391.5 ng/ul, A260/280 =2.17 and A260/230 = 2.35. Then I made first
strand cDNA using Invitrogen's cloned AMV first strand cDNA synthesis kit
according to the instrucrtuions given there, using 3 ul of RNA as
template.I used oligo-dT primer. I tried to PCR out some cDNAs of CYPs
from this, but I got no bands. My positive control also didn't work, so
there is no DNA in the cDNA. What might have gone wrong? Do you think I
should use more RNA as template? I noticed that everyone else in the
literature ( at least for C elegans) uses random hexamers for priming, can this be the problem?
Please give me your input based on your experience with RNA. I have only
a little RNA and don't want to waste it in troubleshooting experiments.
I remember the 260/280 ratio of pure RNA is 2.0, how could it be over 2.0?
And what's your positive control?
bluesoul on Feb 19 2010, 10:12 AM said:
I often find that my total RNA 260/280 is over 2.0. It still works for RT-PCR as long as it looks good on a gel.
To the OP:
How does your RNA look on a gel? You should see strong rRNA bands of the appropriate size(s) (the size varies depending on organism, not sure what it should be for C. elegans).
Also, you need a positive control TEMPLATE in your PCR as well as positive control PRIMERS. You can't conclude from your results whether your taq (or other PCR reagents) went bad or if your RNA went bad. If you can show that both primer sets work on genomic or other positive control template but fail on your cDNA then yes, you have some problem with your RNA or cDNA.
As for how much RNA to use, I have noticed that I will often get a better result from my RT reaction (stronger bands, better amplification plot in quantitative PCR) if I use less RNA. This is probably because sometimes there are PCR inhibitors left over from a TRIZOL reaction. I typically use 500ng to 1ug total RNA (most RT reactions say you can use up to 5ug but I don't usually do this).
I also dilute my cDNA before running the RT-PCR reaction. Usually 3-5 fold (so if the reaction is 20uL I will add 40uL-100uL of water/buffer).
I would be shocked if "everyone" uses random hexamers for RT-priming in C. elegans. If you are trying to get an mRNA out you really want to use oligo dT not random hexamers. I don't know what "CYPs" are but obviously you need to use the appropriate primers to the RNA you are trying to amplify. At any rate, I presume that your control primers amplify a polyadenylated gene so oligo dT cDNA should work for that yes? If you are using rRNA for a control gene then you should use random hexamers.