Arabidopsis Loves

METHOD FOR RNA EXTRACTION FROM Arabidopsis LEAVES (using TRIzol)

The method of RNA extraction using TRIzol is beneficial where it is impractical to separate cytoplasmic RNA from nuclear RNA, microRNAs or endogenous RNases.TRIzol (or TRI Reagent) is a monophasic solution of phenol/ guanidinium isothiocyanate and solubilises biological material while simultaneously denaturing the protein.(Donald C. Rio et al)

SAMPLE PREPARATION(Homogenisation, disruption)/RNA PRECIPITATION*

The samples(watered-control,drought stressed-target) were ground in liquid nitrogen into a fine powder in a clean mortar and pestle. EB **(1.2 mL) was added before the sample thawed.The homogenate,after mixing was transferred into a 2-mL Eppendorf tube, vortexed vigorously

TRIZOL allows clean transfer of the RNA(colourless aqueous phase)by locking proteins, nucleases into the interphase and organic phase(red),(Rio et al).Chloroform (200 μL) was added, and mixed by vigorous shaking for 15 s.The mixture was incubated for 2 min, centrifuged for 15 min at 11 000 × g (4°C). The upper aqueous phase was transferred to an RNase-free 1.5-mL Eppendorf,with addition-mixing of 500 μL isopropanol, and10 min incubation. The supernatant was discarded. The RNA pellet(dry) was then dissolved in 100 μL DEPC-treated, sterilized ddH2O, and mixed with 10 μL DEPCtreated 3 M sodium acetate (pH 5.2) and 250 μL RNase-free 100% ethanol.This was followed by a 20 min incubation and 15 min centrifugation at 11 000 × g at 4°C. The pellet was washed with 1.2 mL 75% ethanol, followed by centrifugation at 11 000 × g at 4°C for 10 min.The supernatant was discarded. The RNA pellet on drying was then dissolved in 50 μL DEPC-treated, sterilized ddH2O, and stored at -80°C then

2.Gel electrophoresis

3.Microfluidics capillary electrophoresis (e.g. 2100 Bioanalyzer)

FRACTIONATION-

. For RT-PCR- Preparation of cDNA or Poly(A)+RNA (eukaryotes) by purification using oligonucleotidecolumn/ streptavidin beads respectively.

REFERENCES

• Chomczynski, P., and Sacchi, N. (1987) Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 162, 156-159

• Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen(2010) RNA: A Laboratory Manual,CSHL Press, Cold Spring Harbor, NY, USA,

• *Ling Meng and Lewis Feldman (2010) A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds. Biotechnol J 5:183-186

• RNeasy Mini Handbook,March 2013 Qiagen.com, (2015). miRNeasy Mini Handbook - (EN) - QIAGEN. [online] Available at: https://www.qiagen.com/gb/resources/resourcedetail?id=632801fb-abc5-4e62-b954-ff51f126a34f&lang=en [Accessed 27 Oct. 2015].

• Thermofischer manual 2015 https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent

APPENDIX

**EB- Extraction buffer