PCR problem

Nov 16, 2015
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#1
I have a problem with my PCRs. From rat liver I have isolated total RNA, then synthesized cDNA with oligoDT primer. After that I used primers for PPARa and PPARb, ERa and ERb for my PCR using also primers for GAPDH as my reference gene. After the PCR my electroforesis in 2% agarose showed clear bands only for GAPDH. This means I guess that my technique is ok and my cDNA good but shouldn't I also see bands for all of my genes since in liver from bibliography all of them are expressed? I have tried it so many times, also with new primers, but the result keeps the same.
 

Ivan

Administrator
Staff member
Nov 16, 2015
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#2
Hi Petroula,



There are a number of reasons why this may not have worked. The fact that the GAPDH PCR worked shows that everything is working, so that leaves your primers as the only other part of your experiment that may not be working. Here are some suggestions as to what could be going on:



1. The genes you are looking at are not expressed in your sample, at least not at a level high enough for detection (GAPDH expression is relatively high so that explains why you are detecting it).



2. Your primers may be good, but the primer design may be bad. If these assays have not been validated my recommendation is to validate them (using genomic DNA if possible) to make sure they work well before using them in experiments like this one.



3. Prepare a second RNA, just in case your liver RNA is deficient in a number of genes you are interested in. While it unlikely that all your genes are not expressed in liver, it is possible so having another source of RNA could help.



Good luck
 
Nov 16, 2015
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#3
Hi Ivan, thank you very much for your immediate reply,

1) I have discussed this many times with many people that have worked on this exact project or with these genes before and everyone said that my genes are highly expressed on liver.



2)I guess the design is good because the PPARs at least, have worked on my co-students. The ERa and -b are new and may have been a mistake in their design. We used injection H2O to dillute them 10pmol/μl as the polymerase protocol suggested. As regards to validating them with genomic DNA, it is extremely difficult because of financial problems in our univercity and unfortunately we dont have materials for DNA extraction in our lab, only for RNA extraction. My total RNA was of good quality beacuse we had two clear bands of 18SRNA and 28SRNA in electrophoresis when I tested it.



3)I already tried with a new RNA from liver of another rat and the result was the same, except one time that with PPAR a had a band in my cDNA from new RNA but also in my older cDNA. I tried also with another tissue and they didn't work.



As you can see something is going really bad but I cant find it. At first I thought that I was doing something wrong, that I am worthless. I use the manufacturer's protocol of Taq and I have tried it so many times that I have eliminated mistakes. I 've tested concentrations on my every material and buffer.

If there is something else you can think let me know. THANK YOU VERY MUCH!!!!


 

Ivan

Administrator
Staff member
Nov 16, 2015
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#4
The first thing I would recommend is not to think that you are the source of the problem. From what you describe you are doing what needs to be done to get this to work.



My recommendation will be to stop trying to force this experiment to work and instead take a step back and try and see if you can get a related experiment to work. In theory your primers should amplify from pure DNA. If you cannot extract DNA maybe you can simply buy some purified DNA. The reason why I recommend this is because purified DNA is cheap, and running a PCR reaction starting with DNA is as simple as it gets. If you cannot get this PCR to work with pure commercial DNA then there is a problem with your primers. In that case then you will have to either redesign the PCRs or get new assays.



Best


 
Nov 16, 2015
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#5
Thank you very much again for your advise Ivan, I am very dissapointed as you can see, but I will fight it! I ll try to ask from my professor to buy a commercial DNA to test the primers, I hope he will agree!!