Controls for RT-PCR reactions - (Oct/19/2012 )
I want to run RT-PCR for some leukemia fusion transcripts including BCR-ABL. I have designed my RT-PCR primers using NCBI-Primer BLAST (I hope this tool is reliable!). My question is:
What should I use as positive and negative controls in my RT reactions? Should I use any genes such as beta-actin or GAPDH? From what i understood after reading this article is that using beta-actin can be a little risky at times. What are your thoughts in using some 'house-keeping genes' as control and which one is the best?
Just to clarify beta-actin and GAPDH are not positive controls! A positive control would be for your gene of interest we have used plasmids containing our gene of interest to perform absolute quantification, this is considered the gold standard but is time consuming.
The genes you are talking about, GAPDH and beta actin are often used as house keeping genes ie they allow for normalisation but ARE NOT POSITIVE CONTROLS! You need to understand this, otherwise your experimental design is flawed!
To establish which housekeeper to use you should look a the expression of these genes during the course of your experiment, if they change they are unsuitable if they remain constant they will suffice.
I think that the paper mentioned is not about real-time PCR. And if Neanderthal is going to do classic RT-PCR (visualise on gel etc.) he indeed needs positive control for his detection reaction, not a reference.
Because in case the RT fails for some reason, the sample would be false negative which can have diagnostic consequences. So some other gene, (any gene, that is always present so basically often housekeepings used also for qPCR normalisation) has to be run on the same cDNA to test, if the RT didn't failed.
Also every group of RT-PCR reactions run together from the same mix should have a positive control for Bcr-Abl included to check whether in this one specific reaction mix, something wasn't ommited (like primers).
But the paper linked is pretty old. For diagnostic detection (in which case you definitelly need positive control) from patient blood samples, the single-step PCR is usually not sensitive enough, so nested PCR was regulary used (my colleague was actually doing this). At least if you trying to monitor residual disease and not just detectiong samples with high percentage of Bcr-Abl.
Nowadays for quantification and detection of low amounts of fusion transcript, real-time PCR is exclusively used. And as far as I know, the reference gene in this case is wild-type Abl, the amount of fusion transcript is expressed as a ratio of Bcr-Abl to Abl.
It would be best if you could specify what exactly are you after with Bcr-Abl.