SDS-TRIzol Combo RNA isolation from high carbohydrate containing tissues

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Introduction

For simple tissues, I prefer to isolate RNA with the Citrate-Citric Acid method, but the isolation of good quality RNA of fruits and other plant tissue with a lot of sugars and/or other inhibiting crap in it can be quite a challenge. The TRIzol method is not always the best option because these compounds sometimes have similar biochemical characteristics as the nucleic acids we try to isolate. In the TRIzol method I have described a less stringent precipitation to get a little less crap, and another way I got rid of sugars was just a freeze thaw cycle of my RNA in water and then a 10 minute spin to pellet whatever was not solubilized. This worked great for Northern blots, and qRT-PCR, but for RNA seq there are all these quality control hoops we have to jump through, something with RNA quality number >7.5 or something artificial you can only determine with an expensive machine, and the quality is then just not good enough. To be honest with you, I believe that all these hoops are in most cases a better safe than sorry approach, and companies jump right in selling us fail safe kits and methods, and machines to check all this. IN the plant field we are already fucked because there is always a special plant kit needed for these weird cell wall containing organisms, and very often the service labs are warning beforehand that they can not guarantee us good results because of plants. But thats for another discussion.

When I needed super quality RNA for RNA seq, I spend weeks using Hot Borate (Gudenschwager et al., 2012), because this was the method they were using in my new lab for high sugar containing tissues, with success for good quality RNA for RNA seq. But for me, it was not the best. First of all the whole method took me 2 days, and I could only do 6 samples at the time because we were doing it in 30 ml oak ridge tubes. I just had degradation and bad RNA quality numbers. I wasted a lot of time, and I missed my phenol based methods, for confidence that I would not have any breakdown. Fran told me already before I started all this that I should use her method she used on maize aleurones (Reyes et al., 2011) based on Holding et al., (2007), that should be good enough. So I tried that method on the last little bit of tissue I had, and it worked perfectly. Important advantages were, time: it took me about 2-3 h, which most of the time was grinding, space: I could do it all in eppendorf tubes and therefore had no problem doing 18 samples at once (in those 2-3h), tissue used: I only used about 0.2 g, and this was enough for a high enough yield for RNA seq, and finally the RNA quality: my numbers were all above 8!!

So try it out, and suck it to the kit selling companies making us believe their mysterious kits with unknown tricks are the only way to isolate great RNA! Good luck!

method (based on Holding et al., 2007 and Reyes et al., 2011)

  • Grind 0.1-0.2 g tissue to a fine powder in LN2
  • Add 200 ul NTES buffer and 200 ul Buffer Saturated Phenol (pH 8):CHCl3 and thaw while mixing thoroughly
  • Once in the tube, spin 10′ @ 4C, @ 10k x g
  • Add another 200 ul Phenol:CHCl3, shake well to mix and spin again 10′ as above
  • Add 200 ul CHCl3 shake it well and place on ice for 5 min, mix halfway by inverting
  • Spin 10′ as above
  • Take the aqueous phase (upper) to a new tube
  • Add 1 mL TRIzol
  • Shake well for 15″ and incubate 5 min at RT
  • Add 200 ul CHCl3, shake well and incubate 2-3 min at RT
  • Spin 10′ like above
  • Take the upper aqueous phase to a new tube
  • Add 500 ul isopropanol, mix well and incubate 10 min on ice
  • Spin 10′ like above
  • Remove supernatant and wash pellet with 70% EtOH
  • Dry pellet and take up in 50 ul RNAse free MQ
  • Check quality of RNA on gel and estimate conc. by spectrophotometer

NTES buffer 50 ml:
20 mM Tris, pH 8 = 1 mL 1 M Tris in 50 mL
100 mM NaCl = 1 mL 5 M NaCl in 50 mL
10 mM EDTA = 1 mL 0.5 M EDTA in 50 mL
1 % SDS = 2.5 ml 20% SDS in 50 mL

TRIzol (concentration):
Phenol (not buffer saturated, pure phenol from crystals) (38%)
Guanidine Thiocyanate 118.16 g/mol (0.8 M)
Ammonium Thiocyanate 79.12 g/mol (0.4 M)
3 M NaAc pH 5 (0.1 M)
Glycerol (5%)

references

Gudenschwager O, González-Agüero M, Defilippi BG: A general method for high-quality RNA isolation from metabolite-rich fruits. S Afr J Bot 2012, 83:186-192.

Holding D.R., Otegui M.S., Li B., Meeley R.B., Dam T., Hunter B.G., Jung R., Larkins B.A. (2007). The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation. Plant Cell 19: 2569–2582.

Reyes F.C., Chung T., Holding D., Jung R., Vierstra R., Otegui M.S. (2011). Delivery of prolamins to the protein storage vacuole in maize aleurone cells. Plant Cell 23: 769–784.

Posted on 12 March, 2014, in lab and tagged , , , . Bookmark the permalink. 4 Comments.

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