We are just starting to do real time RT-PCR and I have question about controls for real time RT-PCR. Do you always do a no RT control for every RNA sample that has been independently extracted and from which cDNA has been prepared cDNA? I am going to do a no template control for sure. The real time will be done using probe based 5' nuclease assay and not SYBR green. The primers and probes were designed using IDT DNA's software and they do not span an exon-exon junction. I have 4 different genotypes to test and am planning on doing at least 2 biological replicates. That means if I include a no RT for each sample, I have 16 samples that will teach then have to be tested for gene specific and a control (housekeeping gene).
Thanks,
Pavithra
Hey,
i normally do one sample no-RT for each cDNA synthesis run but, I do all RNA extraction together. Theoretically it won't make difference if you do everything exactly the same. But, for piced of mind, I'd suggest randomly choosing a couple of no-RT during different RNA extraction runs from different patches.
Hello.
I was doing RNA isolation and RT-PCR in bulk (meaning, a lot of samples in one isolation and RT-PCR) and always included a negative (no-RT) control.
And when doing qPCR remember to include this no-RT control. And don't forget about NTCs in qPCR.
Regards.
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