Can RNA extraction purity affect our target gene expression results on RT-PCR?

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Stephen Andrew Bustin

Purity affects expression in so far as that inhibition can prevent RT and PCR. Also, inhibition is different for different markers.

Richard Bruce McQualter

If the reference gene is amplifying well but your GOI is not, it is probably a primer issue. That being said, you need good quality RNA for qPCR, particularly if you do a one step qPCR. Use a RNA kit to isolate your RNA. Otherwise inhibitors will affect the efficiency of cDNA synthesis and produce a lot of variability between samples.

This site might help: http://www.lifetechnologies.com/au/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting.html

Shambhuprasad T.k.

Both the RNA purity and intergrity measured by RIN (RNA intergrity number) affect the final outcome. If feasible check the RIN its recommended it should be above 7 and 10 being the best.