Low purity in plant leaf RNA extraction and interference from polysaccharides and polyphenols: what should be done?

For procurement validation, it is recommended to aliquot the same batch of poplar leaf powder into multiple tubes and compare 2–3 domestic polysaccharide- and polyphenol-rich plant RNA kits in parallel. Focus on recording three indicators: 260/230, total RNA yield, and qPCR reference gene Ct; do not look only at 260/280. Ask clearly whether DNase I is included, whether β-mercaptoethanol needs to be added separately, and whether there are cases for this plant type. Regarding lead time, domestic reagent suppliers with items in stock can generally deliver within one week. If the budget is within 10 yuan per use, it is recommended to choose a 50-reaction boxed kit or a large package divided into aliquots. If the sales representative is unwilling to provide a trial sample, you can request a quality inspection report for a recent batch.

Sample pre-treatment is more critical than changing the kit. 100 mg of leaf tissue is a bit too much for column-based methods; it is recommended to reduce it to 50-80 mg. After grinding in liquid nitrogen, do not wait for it to thaw; add the powder directly to lysis buffer containing 2% β-mercaptoethanol. PVPP or PVP40 (about 10-20 mg/g tissue) can be mixed in during grinding to adsorb polyphenols and prevent browning. After centrifugation, take the supernatant before proceeding to the column. For samples with high polysaccharide content such as poplar leaves, an additional chloroform:isoamyl alcohol extraction can significantly improve the 260/230 ratio. This modification is very low-cost, and it is recommended to try it on one batch first.

For polysaccharide- and polyphenol-rich plant leaves, it is recommended to prioritize domestic specialized kits, such as Tiangen RNAprep Pure Polysaccharide Polyphenol Plant RNA Extraction Kit, Vazyme FastPure Plant Total RNA Isolation Kit (Polysaccharide Polyphenol Type), and Magen HiPure Plant RNA Mini Kit. When selecting, do not only look at Arabidopsis data; ask the manufacturer to provide 260/230 references for poplar or old grape leaves. At less than 10 yuan per reaction, domestic 50-reaction boxed kits can usually be negotiated, and most have stock with a 3-5 day lead time. Before purchasing, confirm whether the lysis buffer requires additional β-mercaptoethanol, and it is best to choose a model whose instructions explicitly state suitability for polysaccharide- and polyphenol-rich leaves.

If the budget is tight and you don't want to replace the kit, you can modify it yourself into a CTAB method + column purification. Use 2% CTAB lysis buffer with 2% β-mercaptoethanol, lyse at 65°C for 15 minutes, extract with chloroform:isoamyl alcohol and take the supernatant, add 0.5 volume of ethanol and pass through the existing column. This method works well for removing polysaccharides and polyphenols, and the cost per sample is very low, but there are slightly more steps, and care must be taken to prevent DNA contamination. A high qPCR Ct may also be related to inaccurate RNA quantification. Nanodrop readings can be falsely elevated due to polysaccharide interference. It is recommended to requantify with Qubit and then adjust the template amount, which can often bring the Ct back into the normal range.

The 260/230 ratio is only around 1.2, so first rule out residual contamination from the procedure. Confirm that ethanol has been added to the wash buffer as instructed, centrifuge the empty column for 2 minutes after two washes, air-dry at room temperature for 2-3 minutes, preheat the elution buffer to 65°C nuclease-free water before elution, and do not use an elution volume below 40 μL. A high qPCR Ct value is not necessarily due to purity issues; RNA integrity and reverse transcription inhibition can also affect it. It is recommended to run 1% agarose gel electrophoresis to check the 28S/18S ratio, perform on-column DNase I digestion in parallel, and then fix the RNA input amount for internal control. If the internal control Ct is still high, then it is not too late to switch kits.