problems with PCR primers - (Jun/05/2007 )
I purified RNA from human cancer cells using the phenol chloroform method. These RNA extractions were then converted into cDNA using oligo dt primers. I am currently trying to detect the presence of different genes using PCR but for some reason I get no PCR product with most of the primers. Naturally, I double checked the sequence of the primers and try to do the reverse trancription reaction directly with the primers but still I got no results. Moreover some primers worked for a couple of times and then suddenly stoppd working. On the other hand, few of the primers work everytime with no problems at all. I tried almost everything I could think of, I will be happy to hear some new suggestions from people who encountered that sort of problems
Are you sure that you get RNA? I mean, do you spec it prior to cDNA synthesis? Did you try other methods of extraction? Do you use Trizol or PC without guanidin thiocyanate?
If you are sure that template is no problem, check primers again - are they not specific or building dimers etc. You can use Netprimer to check the primers.
Next - reaction mixes: Did you change all concentrations, dilute/concentrate cDNA, Mg concentration or DSMO?
What cycling conditions do you use?
So much to think of, it would help if you provide more specific information...
did you checked purity and intigrity of you RNA sample before RT-PCR???
First thanks for your replys. I did check the RNA using spec and got nice ~500 ng/microliter concentration of RNA (260/280= 1.7). As I allready mentioned some of the primers worked so I'm pretty sure that The RNA is there ( a control PCR of the RNA without the RT enzyme gave no results so I'm pretty sure that the product are not due to DNA contaminations). I haven't tried any other RNA extraction methods, I figured that since I get some products with specific primers the RNA is O.K, I'm not too familiar with the trizol method though it can be a new interesting direction to check. Do you believe that an extraction without guanidine thiocyanate may improve my results?
I'm pretty sure that the primers are O.K and don't cause dimers (I checked them with 'vector NTI' program). As I already mentioned some of the primers worked for a couple of times and then suddenly stopped working. I am currently ordering new primers for a different region in the RNA.
As for the PCR reaction, I don't believe that this is the problem though I'm currently trying to increase the mg concentration. playing with the cDNA concentration or with the number of cycles didn't give substantial results. Using different taq enzymes and PCR mixtures was prooven unuseful.By the why What is DSMO?
The cycling conditions are: 2 min 95, (40 sec 95, 30 sec 55, 90 sec 72) * 35 cycels, 10 min 72.
How about your housekeeping?
Maybe you can borrow some primers that were proven to work from others to test.. just a little suggestion..
Try using a hot start taq - and don't forget the activation time
My house keeping genes work fine i.e, yielding a nice pcr product. I don't see how a hot start taq can help me get a product.
Then I believe your RNA is okay..
Is the RNA that you used for the previous PCR that worked, same as the one you are using now (which doesn't work)?
I have experienced getting different results (no PCR product, unspecific bands, etc) using different source of RNA which I extracted at different time. Although it's the same repeated experiment but maybe we might have done something different to our cells during manipulation.
I use the same RNA preparations for both the "good" and the "bad" primers, unfortunately, I get results only when using the "good" primers. I am currently trying to change the kit used for the cDNA synthesis
maybe you need to adjust the annealing temperature of the "bad" primers. what are their melting temperatures?