TITLE:  RNA Isolation from Plant Tissue Protocol 12: Hot Acid Phenol Method for Angiosperms
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Sarah Covshoff, Rowan Sage and Julian Hibberd</div><div class = "text-block">This RNA isolation method is a multi-component method involving an initial extraction by hot</div><div class = "text-block">acid phenol and then a purification and DNase treatment using the RNeasy Mini Kit by Qiagen.</div><div class = "text-block"><span>The method described below is a modification of a method described by van Tunen et al.</span><span style = "vertical-align:super;">3</span><span style = "vertical-align:super;">.</span></div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div><div class = "text-block"><span style = "vertical-align:super;">3</span><span> van Tunen, A.J. et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light‐    </span></div><div class = "text-block">   regulated and differential expression of flavonoid genes. The EMBO Journal 7, 1257‐1263 (1988).</div></div>

[STEPS]
?. Add  of saturated acid phenol (pH 4.3) and  of RNA extraction buffer to a 15 ml snap cap tube. Warm tube to  in a fume hood using a heat block.
3 ml
3 ml
65 °C
?. Homogenize tissue in liquid nitrogen using a mortar and pestle.
?. Transfer up to  of ground tissue to a tube containing the phenol-buffer mixture and close the snap cap tube completely (second stop). Immediately mix tube by hand. Vortex until the phases mix and appear cloudy. Keep at  or  depending on whether SDS is precipitating.
1 g
0 Room temperature
65 °C
Use a spatula and funnel chilled in liquid nitrogen to transfer the powdered tissue to the tube.
?. Centrifuge at  for .
?. Transfer aqueous phase to a new 15 ml tube.
?. Add of chloroform:isoamyl (24:1 ratio).  Close the snap cap tube completely (second stop) and mix contents immediately by hand. Then vortex until the phases mix and appear cloudy.
3 ml
?. Centrifuge at  for .
?. Transfer aqueous phase to a new 15 ml tube.
?. Precipitate nucleic acids by gently adding  of isopropanol to the tube followed by gentle inversion to mix the phases. Precipitate for  at .
3 ml
4 °C
?. Centrifuge at  for .
?. Remove the supernatant without dislodging the pellet.
?. Rinse the pellet with  of chilled 70 % ethanol made with RNase-free H2O.
1 ml
?. Transfer pellet and ethanol to a new 1.5 ml tube and invert to wash.
?. Centrifuge at  in a microcentrifuge for  and remove the supernatant.
?. Spin dry the RNA pellet by successive removal of any remaining liquid following  centrifugations at maximum speed in a microcentrifuge. Repeat this process until no liquid is seen when the tube is flicked. Allow the pellet to air dry for  to , depending on the size of the pellet.
Avoid over-drying since this will negatively affect re-suspension in water.
?. While on ice, re-suspend the pellet in  RNase-free H2O using gentle pipetting.
500 µl
Ensure the pellet is completely re-suspended before proceeding to step 17.
?. Add  of  LiCl. Precipitate the RNA overnight at .
500 µl
4 °C
?. Centrifuge at  for  at  in a microcentrifuge.
4 °C
?. Wash the pellet three times with  of chilled 70 % ethanol diluted in RNase-free H2O. Centrifuge at .
200 µl
4 °C
Remove the pellet from the wall of the tube to wash the pellet more completely. Pellet will now dislodge easily and care must be taken when removing the supernatant between washes.
?. Spin the pellet. Remove ethanol and dry as in step 15.   Centrifuge at .
4 °C
?. Dissolve pellet in RNase-free H2O on ice.
The volume of the water is dependent on the size and clarity of the pellet, as well as the viscosity of the re-suspension, but   can be used as a standard starting point.
?. Centrifuge RNA extract for  at .Check concentration and integrity using a NanoDrop 2000 spectrophotometer.
4 °C
?. Purify and DNase treat up to  of RNA on a RNeasy Mini Kit spin column (pink) by Qiagen.
100 µg
Follow the ‘RNA Cleanup’ protocol as published in the RNeasy Handbook, including the on-column DNase digestion with the RNase-Free DNase Set by Qiagen.
?. Elute RNA twice from the column using  of  RNase-free water to a total of .
40 µl
95 °C
80 µl
However, if the mass of RNA applied to the column is less than  , then use only   to elute.
?. Add  of saturated acid phenol (pH 4.3) and  of RNA extraction buffer to a 15 ml snap cap tube.
3 ml
3 ml
?. Transfer up to  of ground tissue to a tube containing the phenol-buffer mixture and close the snap cap tube completely (second stop).
1 g
?. Vortex until the phases mix and appear cloudy.
?. Keep at  or  depending on whether SDS is precipitating.
0 Room temperature
65 °C
?. Add of chloroform:isoamyl (24:1 ratio).
3 ml
?. Close the snap cap tube completely (second stop) and mix contents immediately by hand.
?. Precipitate nucleic acids by gently adding  of isopropanol to the tube followed by gentle inversion to mix the phases.  at .
3 ml
4 °C
?. Spin dry the RNA pellet by successive removal of any remaining liquid following  centrifugations at maximum speed in a microcentrifuge.
?. Allow the pellet to air dry for  to , depending on the size of the pellet.
Avoid over-drying since this will negatively affect re-suspension in water.
?. Add  of  LiCl..
500 µl
4 °C
?. Spin the pellet.
?. Centrifuge at .
4 °C
?. Centrifuge RNA extract for  at .
4 °C
?. Warm tube to  in a fume hood using a heat block.
65 °C
?. Immediately mix tube by hand.
?. Then vortex until the phases mix and appear cloudy.
?. Precipitate for  at .
4 °C
?. Repeat this process until no liquid is seen when the tube is flicked.
?. Precipitate the RNA overnight at .
4 °C
?. Remove supernatant by pipette.
?. Remove ethanol and dry as in step 15.
?. Check concentration and integrity using a NanoDrop 2000 spectrophotometer.