different primer efficiency using cDNA and plasmid/PCR product - (Nov/17/2006 )
I really have a big problem. I have plenty of combination of primers for two gene. First step, optimed primer concentration. Second step, primer efficiency: here is where I have problems. I have really good efficiencies if I use like templete plasmid or pcr product, but not when I use cDNA.
Believe, I have 6 pair of primers per gene, I am starting with that and I read everything, do everything, I used primer3 to design the primers. At the beginning I selected only one region in the mRNA to design the primer. Now I introduce all the sequence to get the best primer for the gene, and then?????
Means that realtime PCR is not useful? Primer3 is not useful? etc.
Ah, I tried different standard curve, dilutions 1/10 and dilutions 1/4.
I told you, I did everything.
Please, some suggestions
Thanks in advance
if it works great till you get to cDNA, then the problem likely lies with your template and not your primers
how good is your RNA?
what primers do you use for the RT reaction?
how do you control template quality?
It sounds like your primers and your PCR work fine so the most likely problem is your cDNA. There are two likely reasons why your cDNA may be preventing you from isolating your gene: 1) your cDNA preparation is faulty; 2) your gene is not highly expressed in the tissue cDNA you are using. A couple of suggestions:
-perform a positive control PCR using your cDNA to ensure the cDNA has been made properly - most RT-PCR kits contain positive control primers you can use to test the quality of your cDNA preparation. These primers amplify an abundant house-keeping gene that should be readily amplified from your cDNA if it has been properly prepared.
-prepare your cDNA differently. Your preparation may be fine, but the way you prepare your cDNA may not encourage your specific cDNA to be created. For example, try an extension temperature of 44C instead of 55C to create your cDNA (least likely suggestion)
Assuming the cDNA preparation is fine
-increase the amount of cDNA you are using in each reaction in order to increase the concentration of the template you are interested in amplifying
-research the global tissue expression of your gene to search for other tissues that may express or express more highly your gene of interest. Your gene is more likely to be amplified from those tissues because they contain more copies of your gene. Your gene may also be more highly expressed in specific areas of those tissues, i.e. hippocampus rather than whole brain.
Well, I used a qiagen kit to perform the RNA isolation. Also qiagen kit for RT.
I will try then to get RNA from different tissues and also I will try to increase RNA concentration in the RT reaction, OK?
I agreee with you, I think is the quality of the cDNA. But I am not sure about the cDNA concentration in the PCR reaction, I get Ct about cycle 21.
Anyway, I´ll try new RNA isolation and I´ll tell you what´s going on.
Thnak you very much and good weekend
I´ll see you in the lab
I once also had problems with a primer pair, that didn't amplify with the same efficiency depending on the initial template. Plasmids worked very good (E=0.9), human genomic DNA at a very low efficiency (E=0.7). I didn't find a final answer, but at the end I found a possible one. There was a SNP in the middle of one of the primers that varies significantly in the population. Although this SNP was 8 bp away from the 3' end of the primer, it possibly has an effect in the very initial PCR cycles. So try checking the sequence of the template with BLAST again.