For extraction of high quality total RNA, all chemicals, water, plastic-ware and glassware used for RNA preparation were RNase-free. Glassware and spatulas were covered in foil and baked at 180°C for at least 12 hours prior to use, while plastic containers, electrophoresis tanks, trays and combs were sprayed with RNase ERASE (ICN) and rinsed with DEPC treated water. Disposable plastic ware (pipette tips and eppendorf tubes) were purchased RNase-free, and autoclaved prior to use. RNase-free barrier tips were used for pipetting. Glass beads/Micro-Dismembrator of 0.4 µm (B. Braun Biotech International) were acid washed by stirring for 10 minutes and soaked overnight. The beads were then washed extensively under running water and then
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rinsed with DEPC treated water, dried in the drying oven and then baked at 180°C overnight in a baking oven.
Centrifugation, vortexing, electrophoresis, optical density (OD) determinations and visualizing RNA in electrophoresis gels were performed using the following:
A centrifuge 5415C Microfuge bench top centrifuge (Eppendorf). A vortex MT19DL Deluxe bench vortex (Chiltern Scientific). DNA grade Agarose (Progen).
A 4054 UV/visible (Pharmacia Biotech) or DU 530 UV/visible (Beckman) spectrophotometers.
A UV/visible Darkroom (Pathtech Pty Ltd) connected to Lawork analysis software and Digital Graphic printer Up-D890 and Intelligent Dark box II.
3.3.1.1 Total RNA extraction from S. cerevisiae
Frozen cell pellets from section 3.2.3.4 were thawed on ice and the number of viable cells ml-1 in each sample was calculated. Cells were resuspended in RNA buffer to cell density of 2 x 108 viable cells ml-1. Total RNA was extracted from S. cerevisiae using the glass bead extraction method, essentially as described by Ausubel et al., (1997). S. cerevisiae cell pellets of 2 x 108 cells were resuspended in 300 µl (1 x) RNA buffer and added to approximately 300 µl of chilled, acid-washed 0.4 µm glass beads (Sigma). Samples were kept on ice throughout the procedure. The mixture was vortexed for 3 minutes (alternating one minute vortexing with one minute on ice x 3). Samples were centrifuged at 12,000 g for one minute to pellet cell debris and the upper phase transferred to a fresh microfuge tube. The following two extraction methods were used:
Trizol® Reagent (Invitrogen) extraction method: According the manufacture’s instructions, supernatants containing 2 x 108 cells was mixed with 1.0 ml Trizol® Reagent (Invitrogen) and incubated for 5 minutes at room temperature. Chloroform (0.2 ml) was added and mixed by shaking vigorously for 15 seconds and then incubated at room temperature for 2 – 3 minutes. The mixture was centrifuged at 12,000 g for 15 minutes at 4°C. Following centrifugation, the mixture was separated into phases, the upper aqueous phase was transferred to a fresh tube and precipitated by the addition of
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0.5 ml of isopropyl alcohol, incubated at room temperature for 10 minutes and centrifuged at 12,000 g at 4°C. Following the centrifugation the supernatant was discarded and the RNA pellet was washed (2 – 3 times) with 75% (v/v) ethanol. The washing was conducted by carefully resuspending the pellet with ice cold 75% ethanol and then centrifuging at 7,500 g for 5 minutes at 4°C. RNA pellets were air-dried at room temperature in a fume hood and resuspended in 25 µL RNase free water.
Phenol extraction method: Following glass bead extraction, the cell homogenate was centrifuged at 12,000 g for one minute to pellet debris and the supernatant was transferred to a fresh microfuge tube. An equal volume of phenol/choloroform/isoamyl alcohol (25:24:1) (pH 8.0) and 25 µl Tris buffer (0.5 M, pH 8.0) were added to the supernatant and the content was mixed by flicking it for 20 seconds; and then centrifuged for two minutes at 12,000 g to separate the precipitated protein phase. The upper phase was removed to a new microfuge tube and 3 volumes of chilled 100% ethanol and 25 µl sodium acetate (3.0 M) were added to precipitate the RNA. These solutions were mixed and allowed to precipitate for 2 hours at -80°C (or overnight) and centrifuged at 12,000 g for 10 min. Following centrifugation the supernatant was removed and the RNA pellets were washed 2 – 3 times with 500 µl of chilled 75% ethanol, re-centrifuged for 2 minutes at 12,000 g and the supernatant removed. The RNA pellets were air-dried and resuspended in 25 µl of RNase-free water.
To determine the quantity and quantity of RNA, 2 µl of total RNA solution was diluted with 598 µl of DEPC treated water and A260 and A280 values was determined. To test for purity and reproducibility of extraction across different time points RNA was visualized by electrophoresis of total RNA in RNase free 1% non-denaturing agarose gels in 1 x TAE buffer. A volume of 2 µl of ethidium bromide solution (1 g ml-1) was added to agarose gel solutions. RNA samples (2.0 µl) were mixed with 2.0 µl of 6 x gel loading buffer and 4 µl of RNase free water and loaded onto the gel. Electrophoresis was conducted at 60 – 80 voltage typically for 45 – 60 minutes. Gels were viewed on a UV transilluminator and photographed using a UVP Laboratory Products gel documentation system. RNA samples were stored at -80°C.
48 3.3.1.2 DNase Treatment of Total RNA
Prior to cDNA synthesis all total RNA samples were DNase treated to remove any contaminating DNA. To 20 µl of RNA solution, 2 µl of 10 x DNase Buffer and 1.5 µl of DNase I enzyme (Ambion) were added and mixed thoroughly by flicking the tube and then incubated at 37°C for 45 minutes. DNase I enzyme was then inactivated by the addition of 3 µl of DNase Inactivation Reagent each sample was mixed thoroughly with continued intermittent flicking during the 2 min incubation at room temperature. Samples were centrifuged at 12,000 g for 2 min and the supernatant was transferred to a sterile fresh eppendorf tube. To test the purity of DNase treated RNA, as in Section 3.3.1.1, 2.0 µl RNA gel loading buffer was added to 2.0 µl of DNase treated RNA and 4 µl of RNase free water. The RNA was then resolved electrophoretically in a 1% agarose gel. DNase treated RNA samples were stored at -80°C until required.