If not using a kit then I usually use the TRIZOL extraction protocol, it is very reliable but budget for a few practice runs to get used to it and perfect the technique before you use any important tissue. Analysis by nanodrop (or spec) is essential to detect salt, trizol or other contaminations:

1) Lyse Tissue Sample: Remove the plant material from liquid nitrogen and while it is frozen grind it inside the Eppendorf tube. Immediately add 500μl TRIzol® –(Do not allow TRIzol® to freeze )– homogenise thoroughly on ice. Initial amount of TRIzol® can be varied depending on sample size; maximum 1000μl. There should be no visible pieces of leaf if the sample is ground finely enough. Top up TRIzol® to 1000μl (Keep all samples on ice until you have finished grinding the last one).

3) Warm samples up for 2 min at 37°C

4) Incubate samples 5 min at RT

5) Pellet Cellular Debris: Centrifuge 10 min at 4°C at 12000 RPM

The pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA.

6) Collect supernatant (~500μl) and transfer to new ice cold, labelled, 1.5ml Eppendorf tubes.

7) Phase separation: Add 0.2ml of chloroform per ml TRIzol®. Mix by inversion for 30 sec and then Incubate samples at RT for 3min (Do not mix samples during incubation as RNA needs to settle)

8) Centrifuge: 20 min at 4°C at 12000 RPM

Mixture separates into a lower, red, phenol-chloroform phase, an interphase, and a colourless, upper, aqueous phase. RNA is exclusively in the upper, aqueous phase.

9) Collect upper phase: transfer to new ice cold, labelled, 1.5ml

Eppendorf tubes. It is important not to collect the interphase (protein) or the phenol-chloroform (bottom).

10) Precipitate RNA: At this stage, if your initial tissue sample was very small, glycogen can be added to aid precipitation of the RNA: Add 10μl glycogen to each sample.

Add 0.5ml 100% isopropanol per ml of TRIzol® originally used (too much is better than too little), mix by flicking the Eppendorf tube.

Incubate samples for 10 min at RT, then at least 1 hour at -20°C to precipitate the RNA. The extraction will be much more efficient if this incubation step is left over night.

11) Pellet the RNA: Centrifuge 15min at 4°C at 12000 RPM

12) Remove supernatant – use a big tip first and finish with a small tip.

13) Wash pellet with 80% EtOH to remove salts: Add 1ml 80% EtOH per ml of TRIzol® originally used

14) Centrifuge 5min at 4°C at 12000 RPM

15) Remove EtOH – use a big tip first and finish with a small tip

Check that the tube doesn’t smell of chloroform or phenol – repeat steps 13-15 if necessary

16) Centrifuge again for a short burst and ensure all EtOH has been removed with a fine tip.

17) Air dry pellet at RT by placing tubes upside down on clean tissue for 5min. Do not allow to entirely dry out as this will decrease its solubility.

18) Resuspend RNA: Resuspend RNA in 20μl nuclease-free water, mix by pipetting gently. If you expect very low yield of RNA, this can be decreased to 15μl.

If you have a problem resuspending RNA, heat the tubes for 3min at 55°C

RNA is very fragile and should be stored at -70°C

Analyse the concentration of your sample by spectrophotometer or NanoDrop and by agarose gel electrophoresis

Reference

(Chomczynski P & Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal. Biochem. 162:156-9, 1987)

please visit this website:

http://www.mpbio.com/fast_rna_kits.php?country=223

I would recommend the protocol from Lopez-Gomez (R. Lopez-Gomez and M. A. Gomez-Lim, HortScience 27(5):440-442, 1992) that uses LiCl as selective precipitant for RNA.

Extraction buffer is composed by Tris-Borate (150mM), EDTA (50mM), SDS (2%) and 2-ME (0.1%) followed by phenol-chl freshly added. After LiCl precipitation and consequent resuspension, I would recommend a further precipitation step with NaCl and ethanol

Thank You everyone for valuable suggestions.