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).
Denaturing RNA by Agarose Gel Electrophoresis
RNA is fractionised by running through a 1.2% Agarose, 2% formaldehyde gel (120ml gel = 1.44g agarose, 6.96ml formaldehyde, 12ml 10xMOPS, 100ml DEPC treated water; MOPS and formaldehyde added after dissolved agarose has cooled to 65°C). Samples contained 10 mg of total RNA in 20 ml RNA running buffer consisting of 50% deionised formamide, 7% formaldehyde, 0.5mg/ml ethidium bromide, 1mM EDTA, 20% glycerol, 1xMOPS buffer, 0.25%bromophenol blue.
Prior to loading, samples were heated for 10 min at 65°C. Electrophoresis was performed at 5V/cm gel for 2Hrs in a fume hood.
Radioactive Labelling of Probes
Oligo Labelling Buffer (OLB) : Consists of a 2:5:3 ratio of solutions A:B:C
Solution A: 15 ml 20mM dATP, 15 ml 20 mM dCTP, 15 ml 20mM dGTP, 15 ml 20mM dTTP, 18 ml b-mercaptoethanol, 937 ml solution O. Solution O: 1.25mM Tris.Cl pH 8.0, 0.125M MgCl 2
Solution B: 2M Hepes pH 6.6
Solution C: 25 ng/ ml random hexadeoxyribonucleotides.
50ng of DNA probe is denatured in 13ml of dH20 by boiling for ten minutes. It is immediately transferred to ice in order to retain the denaturised state.
5 ml of OLB and 1 ml BSA (10mg/ml) are added on ice and mixed by pipette. 5 ml of radioactive [ a -32P ] dCTP ( ~ 1.85 MBq) with a specific activity of 111TBq/mM and a concentration of 370 MBq/ml (NEN) is then added to the DNA probe. Next 1 ml Klenow fragment (1U/ml, Promega) is added. This reaction is then incubated for 2Hrs at 37°C.
In order to remove unincorporated nucleotides from the probe, it is passed over a sephadex G50 column (Amersham Pharmacia), and collected in an Eppendorf tube. A further 50ml of TE is added to the eluted probe. The radioactive labelled probe is then denatured by boiling the tube for 5 minutes in water, and transferred immediately to ice.
Hybrisation of radio-labeled Probe to DNA Immobilised on Nylon Membranes
Sodium Phosphate Buffer: 68.4ml 1M Na 2HPO 4, 31.6ml 1M NaH 2PO 4 (pH 6.8)
Pre-hybridisation Buffer: 9ml NaPO 4 buffer, 21ml 10% SDS, 60ml 0.5M EDTA, 0.3g BSA
Low stringency wash: 2xSSC, 0.1% SDS
High stringency wash: 0.2xSSC, 0.1%SDS
Membranes are pre-hybridised at 60- 65°C for two hours in Pyrex tubes containing 25ml pre-hybridisation buffer. This is performed in a hybridisation oven containing a rotating spindle (Bachofer). Radiolabeled denatured probe is then added to the Pyrex tube, and returned to the oven. Hybridisation is performed overnight.
The membranes are rinsed with low stringency buffer, and then washed for 20 minutes with 100 ml low stringency buffer in the oven. is was followed by two high stringency washes of 10 minutes each. Following washing, membranes are sealed in cling film. The strength of the hybridisation signal is assessed using a phosphoimager (Molecular dynamics). For re-use, membranes are stripped of probe by subjecting them to two washes of 10 minutes in boiling 0.1% SDS.
Following electrophoresis, the formaldehyde/agarose gel is placed in water for 15 minutes with light shaking. This is followed by two 15-minute rinses in 10xSSC. The next stage in Northern blotting involves the gel being placed upon two pieces of pre-wetted Whatmann 3MM paper, supported over a reservoir of 10xSSC. The ends of the Whatmann 3MM paper are submerged in the 10xSSC solution to form a wick. A piece of nylon membrane (Hybond-NX, Amersham) should be cut to the same size as the gel, and immersed in 10xSSC for 15 minutes. It is then placed on top of the gel, and any air-bubbles present removed by rolling with a glass pipette.
Three further pieces of Whatmann 3MM paper are cut to the size of the gel, pre-soaked in 10xSSC and placed on top of the nylon membrane. A stack of paper towels are then placed on top, and a ~500g weight was placed on the towels. This encourages the transfer of the 10xSSC from the reservoir, to the paper towels, though a capillary action. The gel should be left overnight, to allow the RNA to thoroughly transfer to the membrane. After the northern blotting transfer, RNA is bound to the membrane by UV-crosslinking (1200 m J UV light, Stratagene Stratalinker), or by baking for 2 Hrs at 80°C.