paranormal qpcr amplification activity - melting curves (May/20/2010 )
I have been reading your discussions for quite a long time, and they always helped me. Today I decided to post my own problems, and I would be very happy if any of you could help, me because I am getting desperated!!
as a summary: I have 3 genes to investigate (1reference and 2 targets). I did my standard curves, check amplification efficiency and so on... Now I am checking my samples (which are a mixture of RNA extracted from larvae infected with bacteria; my targets genes are specific for the bacteria), since there is no way to separate it to get a clean bacterial RNA. I do specific retrotranscription (with a mixture of all my primers, since my sample amount is limited) and perform my qPCR, and all my problems are arising...
When I check my melting curves sometimes I get different peaks and sometimes is everything perfect, I always perform the same protocol, nevertheless sometimes I see a perfect peak at the right melting temperature, and sometimes it is a great chaos. What could be affecting my results?
Other thing taht really frustrates me, is that some times my melting temeprature is changing among replicates... although i pipetted it in the same way, and normally my replicates dont have a great variation in Ct.
Can somebody help me? or give some kind of advice?
thank you very much in advance!!
Don't worry it's not paranormal, it's normal. SybrGreen assay usually has such variability especially when you are working with low template concentration / high Ct values or your reaction conditions are not stringent enough. If you want really perfect assay with no optimization then switch to TaqMan probes. If you can't or don't want then you have to optimize your rection conditions. Lower primer concentration or increase annealing temperature or use more stringent master mix.
Variation of PCR product melting temperature deduced from dissociation curve between + / - 1°C is normal as it is affected by many factors which can change between runs (for example salts in template).
I did a temperature gradient for my different primers, and I am using the one that was quite efficient for all my amplicons, since I want to run them together. I will low the primer concetration (in which range should be appropiate?)
thank you again!
50 - 300 nM forward x 50 - 300 nM reverse