I am studying the expression pattern of few genes in tumor samples treated differently.
Following cDNA making and PCR i can clearly see difference in gene expression on gel. However even though i am using the same set of cDNA that i used for PCR i cannot detect significant fold change in expression of these genes in Real time quantification.
Everything is same in the conventional and real time PCR except the real time PCR has a different master mix containing the SYBR green and different polymerase.
Hence what can be the possible reasons for this? Any suggestions to troubleshoot this observation will be highly appreciated.
Thanks
How you can see gene expression on gel , the real time is more accurate in gene expression measurment so you can depend on the result
There is no standard aproved method to study comparative gene expression in gel electrophoresis.
Conventional PCR is an end point PCR and is less sensitive than real time PCR. Reduce cDNA quantity as the quantity you are using might be reaching saturation. Try diluting your template.
Hello Ahmed,
I do agree with you regarding the qPCR being more sensitive as we can quantify the relative gene expression.
However gene expression studies can be done in 2 ways -
First is the semiquantitative approach where after doing conventional PCR ( end point PCR) the samples have to be run on gel and depending on band intensities we can compare the pattern of expression in our samples.
Real time PCR( quantitative approach) is the next step to it where we are not depending on the end point detection on gel but detection occurs when the threshold is crossed.
Hence in relation to it i have observed a clear difference between samples in terms of band intensities on gel but the same is not replicated in QPCR when i am calculating the fold change in expression between the same samples.
Hello Shefali,
Thank You for this suggestion. This might be a possible reason and so i will try and implement the same.
try a non-structure specific dye. SYBR tends to be specific to folded RNA. exchange your master mix with Ribo-dye.
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