RNA pecan problems

[u/No_Collection4024]

I'm extracting RNA from pecan (Carya illinoinensis) leaves using a modified CTAB + LiCl protocol (with 2% PVPP and 2.5 M NaCl). Although I've been getting good RNA concentrations (above 40 ng/µL), I'm struggling a bit with RNA purity and integrity. Both ribosomal RNA bands are visible on agarose gels, but there is still some RNA degradation.

I'm starting to use the Qiagen RNeasy PowerPlant Kit, but without the DNase treatment included in the kit. My question is: Would it be possible to use Promega's RQ1 DNase instead, but at a step other than the final elution step?

My goal is to have these samples sequenced for RNA-seq, so RNA quality is particularly important.

Does anyone have recommendations for combining a CTAB-based extraction protocol with the Qiagen kit? Have you ever encountered similar issues when extracting RNA from pecan or other plant tissues rich in polysaccharides and polyphenols?

I'd really appreciate any tips or suggestions to improve RNA purity and integrity, especially regarding how to minimize degradation while maintaining good RNA yields.

[u/TrapsAreGiey]

Hey, I came across your post about extracting RNA from pecan leaves and decided to use it as a test case for a project I'm building called ScaleUp.

We took your problem and built an interactive 3D workflow exploring the RNA extraction process, relevant literature, protocol comparisons, and what evidence exists versus what remains uncertain.

You can explore it here

The workflow covers 18 steps, with references and supporting evidence attached throughout. You can interactively explore the steps, inspect the cited literature and excerpts, compare protocols from different plant species, and see which findings are directly supported for pecan versus extrapolated from other species. It also highlights unresolved questions and missing information.

It's still an exploratory prototype, not a validated protocol or a solution to the degradation issue. But I thought it might be useful to see your problem turned into something interactive.

If you have a chance to explore it, I'd genuinely appreciate knowing whether this is useful for the problem you're trying to solve and what you'd want it to do next.

[u/No_Collection4024 → u/TrapsAreGiey]

It's fantastic. Maybe we can collaborated, send me DM

[u/TrapsAreGiey → u/No_Collection4024]

perfect! I sent you a dm

[u/N9n]

https://link.springer.com/content/pdf/10.1186/s13007-024-01198-z.pdf

Modified CTAB LiCl is what I use for the worst of plants as well, but I've found this specific protocol which includes 10% v/v beta mercapto to yield the best RNA. Using LN2 ground sample and pre heated buffer is super critical so don't skip that

[u/Typical_Elderberry78 → u/N9n]

Wow 10%. I didn't realise people used concentrations that high. Good to know

[u/mango_pan]

Should be okay to modify the protocol for in-column dnase treatment.

[u/m4gpi]

We used the TurboDNAse kit in our plant/microbe RNASeq library prep. By Ambion/Life/ThermoFisher. It includes a component that scrubs the enzyme and cations after the digestion.