PCR product one day, none the next day - (Aug/30/2011 )
Firstly i would like to inform everyone that this is my first post! Despite this, i have been coming to this site for the past year many a time and found all your responses to problems extremely helpful as i am a master's student and still green behind the ears. Bunch of gurus you guys are.
Anyways here's my problem and i kinda have to start from the beginning:
I am investigating whether a gene (namely growth hormone-releasing hormone receptor) is being expressed by a panel of cell lines. I am using primers that have been previously published for this gene and shown to work. Now, i extract RNA using trizol and i am well aware that this protocol is prone to gDNA contamination. Anywho, i have found a DNase protocol that gives me good results. cDNA conversion is rather uneventful so i wont go into that. When i PCR (sybr fast system) fresh cDNA (prepared on the day) for this gene, i get the product (~550bp). I then store my cDNA at -80 in single-use aliquots to prevent freeze-thaw degradation. The next day however, using the same cDNA as before but that has been frozen and thawed once, i get no PCR product. In addition to this gene, im am looking for one other gene (growth hormone receptor)(size ~450bp), and the PCR for this gene works without fail even after freeze-thaw. Just based on my growth hormone receptor PCR, i can deduce that my cDNA is still intact enough to use, yet my other PCR produces no product. WHAT IS GOING ON? i thought that if cDNA is good for one gene, it should be could for all (not like RNA when every transcript has unique folding etc) but maybe im mistaken?
I am sorry if this post is tough to read once over, but i tried to supply as much info in one post as possible. any help would be GREATLY appreciated.
I've had some cDNA that worked for some genes and not others, even though with other batches of cDNA all the genes work. I'm not really sure why this is the case. It kinda seems like with primers that work really well and genes that are highly expressed, they will PCR amplify even with poorer quality cDNA. Can you try designing different (better?) primers for the gene that's not working?
In regards to storing cDNA, it's more hardy than you think. I've never heard of storing it at -80. I store my cDNA at -20 and freeze/thaw it many times without any degradation. The problem I describe above occurs from immediately after the cDNA is made, it's not a freeze/thaw issue. I've forgotten cDNA in the fridge for several days, and at room temperature overnight, without any problems (although I wouldn't recommend that!).
Thanks for the prompt reply. I have not thought about designing new primers as optimizing a set of primers could be troublesome in itself, or is it easier than i think?
Regarding the current set of primer im using, since you say cDNA is quite hardy, and since i've already shown this set of primers to work (despite the gene being expressed at low levels), surely it should amplify the next day from the same cDNA? As a last ditch effort i will probably design new primers but im unsure as to how this would help as the current set looks perfect on paper (since the current set does span introns, has no hairpin structures, and low energy primer dimers)
Very important question. HOW are you thawing your cDNA? We've solved so many "PCR related" issues that seemed more complex than they are, simply by scrutinizing our handling techniques. We recently did a freeze/thaw study on cDNA, Oligos, DNA and RNA and ended up modifying what we thought were perfectly acceptable thaw practices. The result has been a noticeable reduction in process variability In the process we were quite surprised to find may publications on freeze thaw effects specifically in genomics and proteomics. It can be a significant source of variability on a host of fronts.
FYI, we found the Box Scientific thaw station to be a pretty good cure all. You set samples on it and it circulates ambient air to thaw them faster without heat. Most of the papers say that faster thawing is better, but only within a safe temperature range for the material being thawed. By thawing everything at ambient temperature the latter risk is mitigated. We used some simple tests to confirm optimum thaw times on the unit for any relevant frozen materials. Because it is a fixed platform, we have been able to proceduralize those thaw times. This has definitely helped on the consistency side.
Very late followup, but if I want to thaw something quickly I put it in room temperature water. Usually it's just 1.5 mL tubes so I have the bottom half of a P1000 pipette tip box on my desk with one of these sitting in it (it just fits) and it's filled with water. Water transfers heat way faster than air so tubes will thaw much faster but won't get any warmer than ambient.