I have been conducting a research study investigating the expression of multiple miRNAs in serum samples from healthy individuals and prostate cancer patients. Following total RNA extraction using the Trizol precipitation method, I obtained RNA concentrations of 80-90 ng/µL with a 260/280 ratio of 1.3 and a 260/230 ratio of 0.2.

For cDNA synthesis, I utilized specific stem-loop primers for each miRNA. During expression analysis, I employed a universal forward primer and specific reverse primers for each miRNA. The cDNA templates were diluted 1:10, and primers were used at a half microliter volume after 1:10 dilution. After performing real-time PCR, I ran the PCR products on a 2% agarose gel with a 50 kb ladder.

My primary concern is the presence of non-specific bands with high molecular weights in samples containing template DNA. Moreover, No Template Control (NTC) samples are also showing band formation.

Specific Questions:

1. What could be the potential causes of these non-specific high molecular weight bands?

2. Why are NTC samples generating bands?

3. What optimization strategies would you recommend to improve primer specificity and reduce non-specific amplification, considering the 1:10 dilution of templates and primers?

4. Given the low 260/230 ratio (0.2), could this indicate potential contaminants affecting PCR performance?

I appreciate any insights, troubleshooting suggestions, or methodological recommendations from the research community.