real time PCR protocol - controls? - (Feb/26/2012 )
I've successfully optimized my real-time RT-PCR (Taqman) to get consistent amplification with my known positive samples. I'm still trying to figure out what controls I need before starting work on the unknown samples.
The primary purpose of the PCR is viral detection, but I would like to quantifying viral loads as well.
During the optimization process, I've been using water as well as master mix/RNA minus reverse-transcriptase as negative controls.
My first question: do I need any other controls for proper quality control? I'm using serum samples for viral detection (not gene expression) - do I need an endogenous control at all?
My second question: for viral quantification, it seems like most people do absolute quantification using a standard curve. I am trying to develop a protocol for this here, but I'm definitely time and space-limited - I'd like to avoid viral culturing if at all possible. After extracting RNA from a known positive serum sample, could I first check the concentration using a spectrophotometer, then make standard dilutions (1:10, 1:100, etc), and use those to create a standard curve? If so, I am thinking I have to create a bank of aliquots of these dilutions to keep at -70? I feel like I'm missing something...
I'm sorry the basic questions - definitely still learning!
In diagnostic lab I did my thesis in there was always isolation control. It tested that samples were not contaminated during isolation procedure, I think it was an empty sample of water isolated together with other samples that day.
Also a positive control to test if your mix is OK. NTC always. I'm not sure if you really need RT- control, it's a RNA virus you detect or transcripts of DNA virus? (if DNA, then it's a question why are you not amplifing it's DNA, but that's reason can be detection of the transcriptionally active virus only). Can your RNA have a DNA form? If not I don't see reason for RT- control (but I may be mistaken).
Usually standards for viral RNA detection are created as in vitro (I think) transcribed RNA from expresson vector containing your amplicon (so no actual virus), dilluted (not sure if with some junk RNA as is done with DNA standards, depends how much other RNA can be present in plasma) and stored -70. Reverse transcribed with actual samples. If it's a common virus you can purchase standards. I'm not sure if you can use serum sample quantified with spectrophotometer, it may contain other RNA than your virus.
I was doing DNA viruses, so I only looked at all this, but I often said to myself I'm glad I don't need to mess with RNA standards. Lots of quanĺity testing required, you need to be sure your standards are not degraded in time.
please can i suggest that you have a look at the Acrometrix(R) pages of the Life Technologies website.
For an easy route to validate your assays this might be helpful.
if you need more information then you can contact me directly on