When performing RNA reverse transcription from plant leaves, inhibition often occurs. Which reverse transcriptase has strong resistance to polysaccharides and polyphenols?

To determine whether there are inhibitors in the RNA, a spike-in experiment can be performed: mix your extracted plant RNA with cell line RNA or known good RNA, reverse transcribe them together, and perform qPCR. If the signal of the known RNA is significantly reduced, it indicates that your RNA does contain components that inhibit reverse transcription. This experiment is more targeted than simply switching enzymes and can avoid wasting samples. If inhibition is obvious, prioritize RNA purification; if inhibition is mild, then consider choosing a reverse transcriptase with high inhibitor tolerance or adjusting the reaction system.

For plant leaf RNA purity, a good OD 260/280 ratio does not mean polysaccharides and polyphenols have been completely removed. It is recommended to first perform agarose or native gel electrophoresis to check whether the 28S/18S bands are clear and whether there are high-molecular-weight carbohydrates or carbohydrates retained in the wells; focus on the 260/230 ratio. If it is significantly lower than 1.8, this indicates residual polysaccharides, polyphenols, or salts. Alternatively, take 1-2 ul of RNA, run it on a gel, and then stain it to check whether there is glycoprotein smearing in the wells. A spectrophotometer can mask problems in low-concentration samples and in impurity peaks with ultraviolet absorbance.

For precious samples, it is recommended to include multiple controls in a single run: no-RT controls from extracted RNA, plant reference genes, known positive DNA/cDNA, and different RNA dilutions. If the no-RT control shows a signal, it indicates gDNA contamination. If the positive control works normally but the plant cDNA shows no signal, the inhibition comes from the reverse transcription step or RNA quality. If the signal appears after dilution, it indicates that the inhibitor concentration has decreased. This approach allows rapid identification of the problem and avoids repeatedly wasting leaf samples. When the budget permits, choose enzymes that are tolerant to inhibitors, but they must be used together with purification and controls.

When polysaccharides and polyphenols are severe, a reverse transcriptase tolerant to inhibitors can help, but it is not advisable to pin all your hopes on switching enzymes. You can first use extraction protocols targeting polysaccharides and polyphenols: the CTAB method, modified Trizol with high-salt/chloroform extraction, selective RNA precipitation with LiCl, or a second pass through a purification column. If the RNA has already been extracted but residual contamination is suspected, wash with 75% ethanol 2-3 times, or add 1/10 volume of 3 M NaAc (pH 5.2) and reprecipitate. Performing reverse transcription after purification is often more reliable than simply switching enzymes.

When selecting enzymes, focus on thermostable or engineered M-MLV, which are generally more tolerant to plant inhibitors than standard M-MLV. You can try adding BSA to the reverse transcription system, with a typical final concentration of 0.1-1 μg/μl, as it can adsorb inhibitors such as polysaccharides and polyphenols; you can also reduce the RNA loading amount, because reducing RNA also reduces impurities. The reaction temperature can be increased to 50-55°C, with pre-denaturation at 65°C for 5 minutes. If even the reference gene cannot be amplified, do not increase the enzyme amount further; first troubleshoot the system.