Does the presence or absence of RNase H activity have a significant impact on subsequent qPCR?

Use the same fungal RNA to perform M-MLV H minus and AMV reverse transcription separately. Use qPCR to examine the internal control Ct and amplification curves, and use conventional PCR to examine the product length. Focus on whether high-GC target genes can be stably amplified, not just total cDNA yield. A 10,000 U gradient comparison is sufficient. It is recommended to also set up a no-reverse-transcription control to rule out genomic DNA contamination.

The reaction temperature of AMV is usually between 42-60 ℃, which does help to open the secondary structure of high GC templates, but its RNase H activity is usually strong. Fungal RNA GC is relatively high. If only short segment qPCR is performed, AMV may be acceptable, but high temperature and prolonged reverse transcription may also exacerbate RNA degradation. Suggest comparing Ct values and products on a small scale first.

Regardless of which enzyme is selected, high GC template denaturation at 65-70 ℃ for 5 minutes before reverse transcription and immediate ice bath can usually improve accessibility. If Ct is still high under high temperature conditions of AMV, M-MLV H minus can be attempted and supplemented with betaine or DMSO. The concentration of inhibitors needs gradient validation to avoid affecting the efficiency of subsequent qPCR.

RNase H degrades the RNA in RNA:DNA hybrid chains, with a more pronounced impact on cDNA yield and long-fragment integrity. qPCR typically uses short amplicons, so Ct value differences are sometimes minimal; however, if downstream applications involve cDNA library construction or long-fragment amplification, it is recommended to prioritize RNase H minus M-MLV to reduce uncertainty caused by loss of template integrity.