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. This method was used on maize aleurones (Reyes et al., 2011) based on Holding et al., (2007). You can do it all in eppendorf tubes, use about 0.2 g/sample, which is enough for a high enough yield and quality for RNA seq.

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

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.