I've been performing RNA extraction on cotton petiole tissue for a few months now using the method described in the following paper, a derivative of the typical hot borate method (https://pubmed.ncbi.nlm.nih.gov/35844778/).
Originally, I was using mortars and pestles that had been baked out for tissue homogenization and had no issues with RNA extraction or purity. I pivoted to using a beadbeater for homogenization to improve throughput and saw an improvement in RNA concentration and purity. Seemingly overnight, my extractions stopped yielding RNA. I've switched to an alternative spin column (Lamda Shredder and RNA Spin Columns vs. the columns provided in the Qiagen RNeasy kit) that collaborators have had success with using the same RNA extraction method, but even with troubleshooting have shown that using Qiagen columns does not fix the issue.
All new reagents have been prepared multiple times over. The borate buffer used for extraction is DEPC treated overnight and autoclaved. All mortar and pestles are baked overnight to remove RNases. DTT is added to the borate just before testing. I've used fresh proteinase K, more proteinase K, included incubation steps. Nanodrop results show less than 20ng/uL and no RNA is observed when run on a gel. I've performed the same extraction method with more ideal tissue, like Arabidopsis leaves, but still no RNA was observed.
It seems that since there is no RNA observed on the gel, there must be some issue with RNA binding to the column or getting washed away before the elution step, but I have no idea how/where. The protocol remains unchanged compared to when it was producing successful results. I'm at a loss in terms of what to troubleshoot next -- I feel like I've tried everything. Would running samples from each wash flow-through be worthwhile to observe where RNA is being eluted, or would contamination/presence of other reagents make this impossible? Open to any and all suggestions or questions anyone might have! Thank you for the help!
If the hot borate method for RNA extraction from cotton petiole tissue is no longer working, consider checking for potential issues like reagent degradation, incorrect reagent concentrations, or sample quality changes. Ensure that all steps, especially temperature and timing, are followed precisely. If the issue persists, try fresh reagents, optimize the protocol by adjusting the borate concentration or incubation times, and ensure that the tissue samples are fresh and properly preserved. Testing the protocol with a control sample could help identify the problem source.
That sort of overnight change to not working makes me think that a reagent has degraded or expired. Proteinase K is very temperature-sensitive and will degrade even in storage in a freezer.
Try with a fresh bottle of Proteinase K on Arabidopsis leaves and let us know what you see.
Good luck!
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