Hi all , I am currently working with a hypothetical protein and I wanted to check if the gene encoding the protein is expressed in clinical strains by using quantitative PCR on cDNA synthesized from the RNA of said strains. I performed qPCR on the clinical strains using gene specific primers and 16s RNA primers. I have calculated the fold change using the delta delta CT method and obtained positive results (which would suggest the gene is upregulated and expressed in all strains?). My question is how do i perform a stat analysis on these delta delta CT values to ensure this did not occur by chance? I am new to qpcr so any help would be appreciated :) .
Hi, You can use positive control strain and one negative control strain if you have a doubt about your CT values. Also, you can check the CT values from some samples without adding primers or cDNA. Then you can get better confirmation about your results. You need to have at least a biological duplicate or technical triplicate for stat analysis. You can run the sample in agarose gel if you wanna see an amplification of your target gene visibly.
Dr Nair, to be sure that the expression is not by chance, you need to show the expression of this gene in a fairly large number of test samples in comparison to control samples, followed by Chi Square test to show that the expression is statistically significant in the test group.
Hi Isla Nair ,
I agree with Arghavan Asghari
Biological replicates are ESSENCIAL for statistical analysis. You need to grow your strains (bacteria?) in at least 3 (8 is better) batches (i.e. biological replicates). You need at least duplicate qPCR reactions for each biological replicate to take the mean.
Use t-test for comparison between two groups (n>8) or use REST (n=3) software as recommended by Arghavan Asghari .
Best,
Jacó
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