Extraction of RNA from Plant tissue
Approximately 100mg of plant tissue is placed in to 1.5ml Eppendorf (1/3 rd in volume), and snap frozen in liquid nitrogen. RNA extraction can then be performed by two methods.
RNA extraction - Trizol ® reagent (Invitrogen)
400 ml of Trizol ® is added to the frozen plant tissue, and ground with an electric drill to homogenise the tissue, a further 600 ml of Trizol ® is added to the sample. Following vortexing for 15 secs, samples are incubated at room temperature for 5 min to permit dissociation of nucleoprotein complexes. 200 ml of chloroform is added and shaken by hand for 15 sec. The sample is next centrifuged for 15 min at 4°C. Following Centrifugation (12000 x g) the aqueous phase is transferred to an Eppendorf containing 500 ml of isopropyl alcohol for precipitation. Following a 15 min incubation on ice, the RNA is pelleted by centrifuging for 10 min at 4°C. The RNA pellet is washed in 1ml of 70% ethanol, and centrifuged for five minutes a 7500 x g. the pellet is then air-dried and resuspended in 44 ml of nucleotide free H2O.
RNA extraction - Qiagen RNeasy Plant mini kit extraction
100mg frozen plant tissue is ground with an electric drill to homogenise the tissue, and 450 ml of lysis buffer RLT added to the sample. The sample is vortexed and incubated at room temperature for two minutes. It is then added to a QIAshredder spin column and centrifuged for 2 minutes at maximum speed. The supernatant is removed and added to 225 ml of 100% ethanol. The sample is added to an RNeasy minicolumn and centrifuged for 15 seconds at 10000 rpm (Eppendorf microcentrifuge). 700 ml of wash buffer RW1 is added to the column and the column centrifuged for 15 seconds at 10000 rpm (Eppendorf microcentrifuge). The column was transferred to a new collection tube and 500 ml of wash buffer RPE added. The sample was centrifuged for 15 seconds at 10000 rpm (Eppendorf microcentrifuge). The RPE wash step was repeated. The column was transferred to a new 1.5ml collection tube and RNA is eluted by the addition of 50 ml of RNase-free water to the column, followed by centrifuging for one minute at 10000 rpm (Eppendorf microcentrifuge).
RNA concentration is then analysed by measuring OD260 in a spectrophotometer (IOD = 40ng/ ml)