Facing problem with Glucokinase and Glucose 6 Phosphate gene in PCR and qPCR gene expression. I have design the primers as per table attached. I have try to run gradient PCR temp between 54-64°C for 30 cycle for both gene however there is no band was observed and have also try to run qPCR for both gene at 60°C for 40cycle there is no CT value. I have check NTC for both gene is negative. I even tried to add DMSO into the PCR product still there is no band observed.
Please advice how to troubleshoot with this low expressing gene. Is there any other method should be followed?
Did you Blast your primers/do they match your targets? What material do you use as positive control? Did you measure RNA cc prior to PCR?
Dear Dr Péter Gyarmati
Thank you for the reply. This primer where designed to validate our experiment involving the exposure of glucose in HK-2 cells. There is no positive control as we are optimizing the primer using a cDNA (RNA concentration is 196.41 ng/ul).
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