Another QPCR problem. Please help - (Oct/18/2005 )
Hi everyone.
I am new to this forum. Just started a MSc degree in Biochem.
I am having problems with my qPCR. I am using Light cycler from Roche. I am testing three cell lines which all express two receptors according to literature. After extracting my mRNA and then making my cDNA using Fermentas First Strand cDNA synthesis kit, I amplified my cDNA of the three cell lines with my primers for the two receptors. Got a nice standard curve for both of the primers. After about three weeks, I am having trouble repeating the results of one of the two primers for which I already developed a clean standard curve.
My first question is how would I be able to test if my primers were degraded? Do I send them for sequencing(there is a DNA sequencing lab a floor above our lab so I can get results back in a day)?
OR do I run a polyacrylamide Gel on my oligonucleotides (I have never run a polyacrylamide gel before)?
Secondly, I used the same reagents and ran both primers using the same cDNA templates at the same time and received amplification for the first primer but the second primer formed primer dimers. So my reagents are not contaminated(i.e. SYBR, Taq, BSA, Pure water,MgCl, PCR buffer,dNTPs). This also means that my cDNA is pure enough to give me amplification because I got results from the first primer.
Finally, my negative control without any cDNA for the second primer shows a crossing point at the same time as my template cDNA. I thought the negative control should have a crossing point much later in the cycle. For example if I do a 42 cycle run on my LightCycler, previously my neg control had a crossing point of 33 and my template cDNA for my lowest dilution had a crossing point of 19.
Its only been four weeks since we got the primers, and I really hope I haven't degraded them.
Thanks to anyone who can help and knows something about the LightCycler.
how are you storing your primers?
as to the second primer, that makes the primer-dimers, your negative control has the same Ct as the sample because the fluorescence you are measuring is what you are getting from the primer-dimer formation.
how are your dissociation curves? you should get the same strong peak in your neg ctrl for that 2nd primer as you do for the wells with template, if I am right
I am confused about how you could have gotten a clean standard curve with the second set of primers, and then gotten primer-dimers under exactly the same conditions? I am assuming you have triple-checked the concentrations of your primers to be sure they are appropriate....
I suppose degradation could be a problem, but it seems wierd that they should degrade that rapidly if you are storing them properly
as to the second primer, that makes the primer-dimers, your negative control has the same Ct as the sample because the fluorescence you are measuring is what you are getting from the primer-dimer formation.
how are your dissociation curves? you should get the same strong peak in your neg ctrl for that 2nd primer as you do for the wells with template, if I am right
I am confused about how you could have gotten a clean standard curve with the second set of primers, and then gotten primer-dimers under exactly the same conditions? I am assuming you have triple-checked the concentrations of your primers to be sure they are appropriate....
I suppose degradation could be a problem, but it seems wierd that they should degrade that rapidly if you are storing them properly
I am storing my primers at -20 degree celsius in ddwater. You are right about the dissociation curve for my negative which is very strong. I also ran a gel on my negative control compared to my cDNA templates and found primer dimers in both samples. If there are primer dimers forming in my cDNA templates, can they have a high crossing point at 23 in a 42 cycle run?
Thanks
"primer dimers forming in my cDNA templates"
what do you mean? are you talking about the dimer formation in your sample wells? and, yeah, they can have a Ct of 23; that just means you're getting LOTS of primer dimer, which is also evident by the strong peak in your dissociation, and perhaps you are adding too much primer to the reaction? are you quite certain the primers are as dilute as they were the first time, when you got the std curve to work properly? too much primer favors dimer formation, and such...
have you tried to find primers for your genes that won't make dimers? if you know you get dimers, it's time to scrap it and start over, especially if you are using SYBR green (I do qPCR that way too, and getting your primers squared away is by far the hardest part)
the only strange thing is that it worked before....but if you are sure you are seeing dimers, and everything is the right concentration, then it's time to go back to square 1 with the primer design
i'm sorry, man, that's frustrating
what do you mean? are you talking about the dimer formation in your sample wells? and, yeah, they can have a Ct of 23; that just means you're getting LOTS of primer dimer, which is also evident by the strong peak in your dissociation, and perhaps you are adding too much primer to the reaction? are you quite certain the primers are as dilute as they were the first time, when you got the std curve to work properly? too much primer favors dimer formation, and such...
have you tried to find primers for your genes that won't make dimers? if you know you get dimers, it's time to scrap it and start over, especially if you are using SYBR green (I do qPCR that way too, and getting your primers squared away is by far the hardest part)
the only strange thing is that it worked before....but if you are sure you are seeing dimers, and everything is the right concentration, then it's time to go back to square 1 with the primer design
i'm sorry, man, that's frustrating
Thanks so much for your help. I am going to re-check my concentrations for primers. I started with 10pmol/ul first which some people in my lab said was too high. How about lowering it to 4pmol/ul? Maybe that would help me. Its just hard for me because it worked in my first try but the subsequent reactions have been crappy.
I usually design my primers with Primer Premier 5.0. The sense/antisense sequences didn't say any dimers would form.
Anyways i am going back to the drawing board and hoping for another better set of primers.
Later.
I have found good qPCR primer design to be a big PIA and the only part of the whole technology that is really hard. I use Primer Express or that other free one on Invitrogen's web site, but then I always Blast them myself, do the intron/exon analysis, check for secondary structure and all that stuff by hand.
I would try a gradient of primer concentration with some cDNA or gDNA stock, to see what concentration of primers is optimal, before ever generating a standard curve. this is an important step. I tried to skip it when I started with qPCR and just did a rough version, well two of my 5 genes had poor efficiency and I ended up having to go back anyways. And sometimes, a little more of one primer and a little less of another is works better too.
good luck