We are thinking to manually isolate RNA using trizol or a reagent like 'Plant RNA reagent' which even uses a simpler protocol and yields high quality RNA. Please check this link:
http://tools.lifetechnologies.com/content/sfs/manuals/plantrna_man.pdf
We want to extract total RNA for doing RNA-seq, small RNA-seq and degradome sequencing (PARE), so we don't want any size-fractionation and use the same RNA for all sequencing purposes. Shall we necessarily use kits? We will check the quality of RNA on Urea gel before sending the samples to the facilities for above sequencing and the quality will be subsequently checked on bioanalyzer before proceeding further. So if the quality is good the isolation procedure doesn't matter, isn't it? Manual methods give high yield and remove the hassles of size fractionation. Will the presence of some genomic DNA contamination hamper the process? Are manual methods not better than the kits to get rid of genomic DNA or yielding high quality RNA?
Any suggestions?
Thank you.
It is not advised to use any column-based purification methods if you want to sequence small RNAs. The columns do not bind smaller length RNAs very efficiently. Not sure what the best method for extracting plant RNA (my experience is with bacteria), but trizol should do the trick nicely, I believe you should freeze plant material with nitrogen and grind with mortar & pestle before trizol extraction, with ethanol precipitation to recover the RNA.
Little to no point in analysing the total RNA on a urea gel if you are using the bioanalyser to assess quality - the bioanalyser is like a gel, except you have much higher resolution of the rRNAs, just ensure that your 260/280 ratios indicate good clean RNA, the RIN is higher than 8.5 (guideline) and there is no contaminating gDNA - it is visible in the bioanalyser output as a 'lump' running above the rRNA (check by PCR).
gDNA contamination will definately cause a lot of artifacts in the RNAseq results, it will be incorporated into the RNAseq libraries during the shearing and primer ligation steps, best to get rid of as much as you can by DNAse digest (followed by repurification), again use off-column methods to preserve your small RNAs.
I was using a very basic cold acid phenol-chloroform method and obtained consistently clean and pure RNA with RIN > 8.8. The low pH of the phenol helped remove a lot of the gDNA before DNAse digest.
Yes. Some use trizol, or buffer with CTAB. The isopropanol overnigth help in get more RNA.
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