I completed the extraction of RNA from colon samples to measure the relative gene expression level of genes related to the immune response, but the same reason each time even with the master mix changing when performing samples on RT-PCR, the attempts were unsuccessful and the florescence was not more than 150 RFU, although that genes on the agarose gel is perfect and identical to its size.

hopefully if this was happens with someone, and a solution has been found, give us suitable and optimal option to fix that malfunction.