How many -RT Controls do I need - (Oct/25/2007 )
A little bit of background so the question makes some sense. I micro-dissect wings and legs from three different strains of my model organism (Nasonia) and then test levels of two different genes in each tissue. That gives me 2tissuex3strainsx3genes(2+RP49 control). I am trying to fit them onto a single 96-well PCR plate and still have room for -RT controls.
1. Do I need to run a -RT control for each of the genes, or can I just have one row of -RT controls? i.e. -RT controls for each tissue from each strain.
2. I run the samples in triplicate, do I need to do the same for the -RT controls or can I run them in say, duplicates?
Thanks for all the help!!
I would say you could get away with duplicates, but I would definitlely do them for each gene. usually my triplicates all look the same but sometimes there is one that peaks a little at high cycles while the rest are clean.
Each gene. Duplicates. (I even do only one well -RT sometimes. Baaad practice )
I am new at this (just starting out) but I was wondering: is it acceptable to only test for a housekeeping gene in your -RT controls?
I am asking this since I will be doing a large RT-PCR experiment with 27 genes, so this would save me an enormous amount of time, money and tissue sample.
I would first prepare all cDNA samples before amplificating the 27 genes of interest so the samples can't become contaminated with amplicon (these genes have never been studied before in my lab so I believe there is no chance of them contaminating samples or having contaminated lab material in large quantities). My reasoning is: if there is a contamination of my samples, this contamination should include common housekeeping genes, so checking for contamination with housekeeping genes is sufficient.
Is my reasoning correct or should I test all 27 genes?