Interpretation of Ct values - (Dec/04/2010 )
I am studying the expression of phosphate regulatory genes of Rhizobium using Real time PCR. I performed the Real time PCR using SYBR green kit and ABI 7500 PCR system. After normalization of the data, I got these Ct values
1. Endogenous control (16S rRNA) Ct: 13.25
2. Gene 1: phoB: 12.42
3. Gene 2 : phoC: 12.11
4. Gene 3: phoA: 14.64
I learnt from the Real time PCR manual that the Ct value is a measure of the abundance of the gene/ mRNA. Lower Ct values mean high abundance and higher Ct means low abundance
Does this mean that genes 1,2 are expressed at a higher level than that of the endogenous control?
and gene 3 is expressed at a lower level than the endogenous control?
no, without knowing the amplification efficiencies of your assays you cannot compare expression levels between different genes. usually you are also not interested to know if gene A is higher expressed than gene B. most often you want to know how the expression level of your GOI changes within different (treated) samples.
Your Ct values are quite low. Make a standard curve for each amplicon using different dilutions of your template cDNA to calculate the different amplification efficiencies and to check whether you are still in the linear range with your low CT values. I assume you are using undiluted cDNA?