Hi all. I am trying to determine if a gene is expressed in clinical strains. I was told to perform qpcr using cDNA synthesized from the RNA of those strains. My question is how do I analyze the data to determine if the gene is actually expressed? and how would I present those results? if I Run a gel using those qpcr products and it’s the correct size does that mean the gene is being expressed? Or do I report on the Ct values and fold change? I am new to qpcr and any help would really be appreciated
thank you in advance
Hi, You have to use a double delta Ct formula to calculate mRNA expression and expressed in fold change if you used sybr green dye for qPCR. you no need to run the gel. you can run the gel if you really wanted to just see the expression. you can present the mRNA expression in fold change. you can trust the expression if you see the band at the correct size. You cant just express the CT values in the result section. you have to substrate the loading control gene CT values from the target gene expression CT values if you do Double delta CT values. Herewith I have attached the model sheet you can use for your stat and understand the calculus.
In the first you must ensure the integrity of RNA extraction by find 2 distinct bands on agarose gel, and you must take the same concentrations for all samples to convert into cDNA for the corrected values.
Use an endogenous control as GAPDH or Beta actin as reference gene.
Insert melting curve at Real time PCR program in the thermal cycler to ensure there is no primer dimer.
Calculate the relative gene expression relative to the control or the starting step of experiment.
The calculation by using M.Pfaffl model 2001, as follows:
A new mathematical model for relative quatification real time RT-PCR,
Mickael M. Pfaffl 2001, nucleic acid research vol.29
Ant thing is not clear do not hesitate to contact me.
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