How to construct a standard curve for real time PCR - (Aug/31/2010 )
I have a big problem here and maybe someone could help me . I want to compare gene expression profiles between rats showing individual differences in several depression-related tests. I have chosen to work with SYBR GREEN in order to reduce the project costs and, because of this, I'm going to use a standard curve for the quantification Real Time PCR (a non-related sample will be retro-transcripted in a gene-specific way using the reverse primer from every specific PCR). At this point, I have several questions:
1. Is there an accurate method for quantifying the gene-specific cDNA using the nanodrop??? (because a direct quantification is not recommended).
2. Once I have the quantified sample, I prepare several dilutions and with those dilutions I construct the curve. Do I have to quantify every dilution or I can use the dilution factor????
What do you think, is this design appropiate???
I really need help!!!!
NanoDrop, or spectrophotometry more generally is just to quantify total DNA or RNA and its quality (or other spectrophotometric properties of samples). I highly doubt it could be used for gene-specific quantification. However, the standard practice is to use the spec to guide diluting the total DNA or RNA concentrations to be equal across samples. As I recall this is usually done before the reverse transcriptase reaction and perhaps again afterwards.
If you are using a standard curve method of quantification, you will want to be sure you have enough sample to use the same standard curve in all reactions. Using the standard curve you can estimate the starting concentrations of each other reaction.
Hope this is helpful. There are a lot of different protocols online which can probably give you helpful details.