TITLE: RNA Extraction Full Protocol
AUTHORS: Onkar Nath, Stephen Fletcher, Alice Hayward, Agnelo Furtado, Robert J Henry, Neena Mitter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA Extraction protocol suitable for all Avocado tissues. This protocol also suits extraction from many other plants like Capsicum, Finger lime and Cotton.</div></div>

[STEPS]
?. [Tissue Lysis]
Finely grind frozen tissue. Grinding in the buffer is not recommended.
?. [Tissue Lysis]
Preparation on the Day of UseAdd  10% SDS per 2ml OB1 buffer and  β-mercaptoethanol (Fume-hood should be used for working with 2ME)Heat the buffer to .
10 µl
65 °C
?. [Tissue Lysis]
Add approximately  (0.01 g) of ground frozen leaf tissue to  hot OB1 buffer in 2 ml tube and incubate at  for .
10 mg
650 µl
65 °C
Over-incubation leads to RNA degradation and delicate tissues for e.g. roots cannot tolerate this. Tissue-specific recommendations are reported in RNA extraction section
?. [Tissue Lysis]
Centrifuge at  (14,000 rpm) for  at , pour supernatant to another tube. This improves the cleaning process.
Centrifuge: 20817 34
0 Room temperature
?. Add an equal volume of OB2 buffer.
?. [Removing Impurity]
Add an equal volume of chloroform isoamyl alcohol (C:IAA); to be performed in fume-hood. Vortex tubes well.
?. [Removing Impurity]
Centrifuge at  (14,000 rpm) for  at .
Centrifuge: 20817 34
0 Room temperature
?. [Removing Impurity]
Collect the supernatant and repeat chloroform wash.
?. [Precipitation]
Add an equal volume of 100% Isopropanol (Cold).
Preferably precipitate with a half volume of   LiCl instead of isopropanol, if the ratio of high molecular weight and low molecular weight RNA does not matter for the experiment.
?. [Precipitation]
10.   Incubate at  for  or longer as needed. Incubating longer increases yield but adds more salt in precipitant.
-20 °C
?. [Precipitation]
11.   Centrifuge at  (10,000 rpm).
Centrifuge: 12857 34, 25 min, 4 10
?. [Precipitation]
12.   Wash pellet with  of  ethanol.
1 mL
?. [Precipitation]
13.   Centrifuge at  (8,500 rpm)
Centrifuge: 7674 34, 10 min, 4 10
?. [Elution]
14.   Air Dry the pellet. Do not over-dry but get rid of maximum ethanol possible.
?. [Elution]
15.   Dissolve in 20 µl of ultrapure DNase RNase free water.
?. [DNA Degradation]
17.   If DNA must be removed from the RNA sample, add 1/20th volume of  and 1/20th volume of .
?. [DNA Degradation]
18.   Incubate at  for .
37 °C
?. [DNase Enzyme removal]
19.   Add water to make up  of total volume.
500 µl
RNAClean XP Bead protocol from Beckman Coulter can also be followed to remove DNase enzyme. TRISure protocol has been followed because of cost factor.
?. [DNase Enzyme removal]
20.   Add an equal volume of
Use OB3 if RNA degrades with the use of TRISure.
?. [DNase Enzyme removal]
21.   Incubate for .
?. [DNase Enzyme removal]
22.   Add  chloroform.
100 µl
?. [DNase Enzyme removal]
23.   Centrifuge at  (14,000 rpm), collect supernatant.
Centrifuge: 20817 34, 20 min, 4 10
?. [DNase Enzyme removal]
24.   Add an equal volume of isopropanol
?. [DNase Enzyme removal]
25.   Incubate at  for  or longer.
-20 °C
?. [DNase Enzyme removal]
26.   Centrifuge at   (14,000 rpm).
Centrifuge: 20817 34, 40 min, 4 10
?. [DNase Enzyme removal]
27.   Ethanol wash pellet.
?. [DNase Enzyme removal]
28.   Dissolve in 20 µl of ultrapure DNase RNase free water or 1x TE buffer as per requirement.
Allow the pellet to dissolve and store at -20 or -80 as required. Do not leave at room temperature or 4°C for long.
?. [Optional clean up and assessment]
30.   Quality-assure using Nanodrop and Quantify using Quantifluor RNA, or Bioanalyzer.
?. [Elution]
Pool tubes if required at this stage for higher RNA content.
?. [Optional clean up and assessment]
Optionally if the pellet is still not clean, follow RNAClean XP Bead protocol from Beckman Coulter. Briefly, add beads to solution, separate beads attached to RNA using magnetic rack, ethanol wash and elute in DNase RNase free water (use 100µl buffer instead of the recommended, 90µl per 50µl sample) (Follow manuscript's RNA Extraction Section for specific recommendations and discussion).