question about RNA concentration for real time PCR - (Dec/02/2013 )
We are getting ready to run some real time PCR and recently read that the same concentration of RNA should be used for each of the samples.
Our chemistry department has an Ultrospec (Amersham Biosciences) spectrophotometer (similar to a nano drop) that has a ultra low volume cuvette (7 uL) and we are using this to quantify the RNA.
My question is this- How is the easiest way to ensure that we use the same concentration of RNA each time? Is it best to dilute the RNA from each sample down to some predetermined concentration that way our students can use the same amount of RNA each time rather than have to take this into account for each sample? I am trying to come up with a way to minimize errors and make it easier for all involved.
If dilution is the answer, is it best to use the elution buffer that we used from our RNA extraction kit (5 Prime Perfect Pure) or is sterile water ok? Thanks in advance.
In the ideal case all samples will be at the same concentration in the same buffer. Most kits allow you to elute with water if you wish to avoid buffer differences. Also the quality of the RNA should be assessed by RNA gel or bioanalyzer. Even when you do all of this there will be some sample to sample variability. This is why you will need some well-chosen reference genes for normalization purposes.