From a population of transfected/transduced cells I sort those with the desired expression. Starting from 10.000 cells I extract the ARN, and from its cDNA I amplify the gene of interest, as well as b-actin as a housekeeping control, by specific primers. However, I only detect b-actin and not the aimed gene. Any feedback about this matter?
Have you used these primers and probes before to detect the target?
Were you able to deduce if the transcription process was successful?
Hello Stephen,
The primers are usually use it for amplify the target, indeed I made a possitive control for the PCR and it works succefully.
The trasncription should have worked because I saw the desired protein exppression by FACS.
If you are certain about those steps and expression of something is happening then it may be the PCR primers are not binding to your product. May be the transcripted product is mismatched somehow. Have you repeated the transfections?
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