rt-qPCR crazy plots! - (Oct/06/2005 )
I am having a lot of probelms running this rt-PCR. I am using DMC primers and COX as a housekeeping gene, both have been optimized already.
I synthesized cDNA (from RNA extrcted with Trizol and purified with promega SV columns; RNA integrity checked in 1% agarose gel) using IMPROM Promega revese transcription system.
This cDNA was diluted 16X and used for rt-qPCR using Finnzymes F-410L Master Mix (SYBR) with OPTICON monitor 2.0. The results are miserable!! I got lines (not curves) that started from cycle 1 and went right ahead, really high fluorescence, but the blanks look OK and the melting points are the expected ones.
I have tried everything already, I've used 2X, 8X diluted cDNA, changed Master mix tube, changed software (from OPTICON 3.0 to 2.0), reset master file, changed sample just to check primes, changed primers, and the results seem to be the same everytime. I've been very careful in manipulation and steriliry. I've already ran rt-qPCR and everything went fine, I dont know what's going on!
I DONT KNOW WHAT TO DO!!!
please ANY comment will help!!!!!
"I've already ran rt-qPCR and everything went fine, I dont know what's going on!"
what do you mean? when was it that it worked? more details, please!
I would assume way too much template(not very likely, you should see something) or a problem with the software/bulb
have you run a background check on the block?
what exactly do you see on your negative controls? (ntc, h2o)
what do you mean your melting curves are OK? apparently you are not getting amplification, you shouldn't see anything but possible primer-dimers on your curves?
can you post a pic of the amplification plot and the dissociation curves from a run, please? I am not sure what you are seeing.