Two RNA extraction methods were used. For initial isolation of genes from
Eucalyptus, a modified hot borate method described by Wilkins and Smart ( 1 996) was used. For gene expression studies the RNeasy plant mini kit (QIAGEN GmbH, Hilden, Germany) was used. For both methods RNase-free components were essential and it was done by baking all glassware at 1 80°C for 6 h; all plastic-ware was soaked in 3% (v/v) hydrogen peroxide for 1 h and rinsed with milli Q water and autoc1aved in aluminium foil; all mortar and pestles were wrapped in aluminium foil and autoc1aved; and all solutions were treated with DEPC @ 1 mllI.
4.2.2. 1 Hot borate RNA extraction
The hot borate extraction (XT) buffer was prepared by adding: • 0.2 M Na borate decahydrated (borax)
• 30 mM EDTA • 2% (w/v) SOS • 1 0 mM dithiotrietol (OTT) • 1 % Nonidet P-40 (NP-40) • 2% (w/v) polyvinylpyrrolidone (PVP) 92
Apical bud
Paired bud retained for visual observation
Axil of the bud excised for gene expression analysis
Figure 4.1 Typical example showing sites of bud collection for meristem identity gene expression analysis. Axillary bud was excised from the position shown above. The paired axillary bud was retained for visual examination to determine if the bud was floral or vegetative.
All the chemicals were dissolved in RNase-free, pre-warmed milli-Q water, the pH was adjusted to 9 with 2 M NaOH, and the stock solution was autoclaved. DTT, PVP, NP- 40 were added just before the time of extraction.
Aliquots ( 1 g) of frozen (-80°C) plant tissue were deep frozen in liquid nitrogen and ground to a powder using a mortar and pestle. The finely ground powder was transferred into a cold Oak Ridge tube and chilled in liquid nitrogen. The XT buffer was heated to 80°C in an Oak Ridge tube. The cold powder was transferred to 5 ml of hot buffer and vortex was done for 30 s, 1 05.5 J-lI of proteinase K (20 mg mrl in water) was added, and the mixture was incubated at 42°C for 1 .5 h with gentle swirling and mixing every 1 0 min. After that, 400 J-lI of 2 M KCI was added and the extraction mix incubated on ice for 30 min with gentle and horizontal swirling. Cellular debris and denatured proteins were pelleted by centrifugation at 26,000 x g for 20 min at 4°C, and
the aqueous supernatant transferred to a fresh tube. RNA was precipitated at 4°C overnight by the addition of a 1/3 volume of 8 M LiCl (to a final concentration of 2 M LiCI), after which the precipitate was pelleted by centrifugation at 26,000 x g for 30 min at 4°C. The supernatant was decanted and discarded.
The pellet was gently dispersed and washed four times in 4 ml of ice cold 2 M LiCl. The samples each time were centrifuged at 1 2,000 x g for 1 0 min at 4°C. The
supernatant was decanted and discarded. After the final wash the pellet was suspended in 2 ml of 1 0 mM (pH 7.5), by warming to room temperature and followed by a gentle vortex. The insoluble materials were pelleted by centrifuging at 1 2000 x g for
1 0 min at 4°C. The supernatant was carefully pipetted to another glass Corex tube and 1 1 1 0 volume 2 M potassium acetate (PH 5 .5) was added, and incubated for 1 5 min on ice. Centrifugation was followed at 1 2,000 x g at 4°C for 1 0 min to remove
polysaccharides and insoluble material. The supematant was transferred to a clean 1 5 ml Corex tube and RNA was precipitated by adding 2 . 5 x volume of 1 00% ethanol and left overnight at -20°e. RNA was pelleted on the next day, by centrifuging for 30 min at 9,000 x g at 4°e. The supernatant containing ethanol was discarded. The RNA pellet
was gently washed with 2 ml cold 70% ethanol and centrifuged at 9800 x g for 5 min at
4°e. The supernatant was removed carefully and also residual ethanol was removed under vacuum. The pellet was re-suspended in 200 III RNase-free water and transferred
to a 1 .S ml tube. The RNA was quantified and stored in aliquots at -20°e. Small aliquots were run on gel to visualise the sharpness of 1 8S and 2SS bands (Section 4.2.7).
4.2.2.2 QIAGEN RNA extraction
A frozen bud sample (20-40 mg) sample was ground in liquid nitrogen. The sample was transferred to 1 .S ml tube. By following the instructions of the manufacturer (QIAGEN), the following method was used. Before starting the extraction 1 0 III � mercaptoethanol was added to 4S0 ml (RLT) lysis buffer (supplied by the
manufacturer), which contained guanidine and isothiocyanate buffer. The frozen ground tissue was added to the lysis buffer and vortex was done vigorously. The lysate was pipetted directly onto a QIAshredder spin column, placed in 2 ml collection tube and was centrifuged for 2 min at 1 4,000 rpm. Supematant of the flow-through fraction was carefully transferred to a new micro centrifuge tube without disturbing the cell debris pellet in the collection tube. Ethanol (96- 1 00%) of O.S volumes was added to the cleared lysate and mixed immediately by pipetting. The sample was applied to an RNeasy mini column, placed in a 2 ml collection tube, the tube gently closed and centrifuged for I S s at 1 0,000 rpm. The flow-through was discarded. DNase treatment was done at this stage as described by the manufacturer (QIAGEN). After washing with RWl buffer (supplied by the manufacturer) the RNeasy column was transferred into a new 2 ml collection tube. RPE buffer (supplied by the manufacturer) of SOO III was pipetted onto the RNeasy column and centrifuged for I S s at 1 0,000 rpm, to wash the column. The flow-through was discarded. The second wash with RPE was done and centrifuged for 2 min at 1 0,000 rpm to dry the RNeasy silica-gel membrane. To eliminate any chance of buffer RPE carryover, RNeasy column was placed in a new 2 ml collection tube and centrifuged at 1 4,000 rpm for 1 min. The RNeasy column was transferred to a new 1 .S ml collection tube to elute. RNase free water (40 Ill) was pipetted directly onto the RNeasy silica gel membrane, and centrifuged for 1 min at
1 0,000 rpm to elute. RNA quality and quantity was measured using the Nano Drop spectrophotometer (NanoDrop Technologies Inc, Wilmington, D.E., U .S .A).