I have got a severe problem when doing the miRNA research using real-time PCR. We have found a target urinary cell-free miRNA showed different expressions in cancer through miRNA microarray test, and evaluated the miRNA’s expression in about 400 urine samples. Real-time PCR results using SYBR green have confirmed the microarray test result. Then we were trying to do a validation test. Because there were no residual primer (ordered from company A) used in the training test left, so we have reordered another newly synthesized primer of this miRNA (from company A). And we continued to do the validation study using the same methods: the same miRNA extraction method, the same cDNA synthesis method, the same quantification method (Real-time PCR, SYBR green). What have changed were the urine samples from different persons (the same cancer) and the primer.

But the copy numbers of the miRNA this time were 100times lower than the former study. What made it different…? 1) urine samples from different persons?- we donot think so, because the extracted urinary miRNA’s concentration were similar, and the copy numbers from real-time PCR were all lowered regardless of cancer patients or non-cancerous patients. 2) the newly synthesized primer? – We have tested for numerous primers from company A and the other company B,C,D, etc. The results were the copy numbers of the miRNA were all different when using different primers…….

Additionally, we have tested for the miRNA extraction kit and cDNA synthesis kit to see whether the copy numbers of the miRNA vary. We used a test set (10 cancer and 10 non-cancerous urine) , to extract miRNAs several times, and synthesized cDNA to do the real-time PCR. The results showed there were no significant differences. Then using the miRNAs extracted from the same day to synthesize cDNA for several times, and did the real-time PCR analysis. The results showed there were no significant differences.

So we move our attention to the primer… But still cannot find the key point of the problem.

Is there anyone having similar experiences? Please give me some advances.

Thank you…