Total RNA Isolation:
To each worm pellet, 4 ml Gibco BRL Trizol was added per ml of packed worms, and vortexed vigorously until completely resuspended (longer than 1 min). This slurry was flash frozen in liquid N2, thawed at 37°C, and then vortexed. The freeze/thaw cycle was repeated, and the mixture stored at -80°C until all samples were collected. After thawing, 2ml Trizol/ml starting packed worms was added, and vortexed vigorously. Per ml of starting packed worms, 2 ml CHCl3 was added, the tubes were shaken by hand for 15 seconds, and then allowed to sit at RT for 3 minutes. The tubes were then centrifuged at 12,000g, for 15 minutes at 4°C. The top aqueous layer was removed to a fresh 15-ml conical tube, taking care not to disturb the interphase. An equal volume of isopropanol was added, and the tubes mixed well, and then allowed to sit at RT for10 minutes. The RNA pellets were then washed in10 ml 75% EtOH, spun at 7500g, for 5 minutes at 4°C. The pellets were briefly air-dried, and then dissolved in DEPC-ddH2O (0.5-1 ml per ml of starting packed worms). The concentration of the RNA was determined by spectrophotometer, and the quality of the RNA was checked by running 1ul on a standard 1% TAE gel.
mRNA Isolation (adapted from Joe DeRisi)
1. The oligo-dT cellulose was prepared by resuspending 1g (1 vial of Ambion oligo-dT cellulose) in 10 ml 1x NETS (100mM NaCl, 10mM Tris-Cl pH7.4, 10mM EDTA, 0.2% SDS), and then washing the cellulose 3 times with 10 ml 1x NETS in a 15-ml conical tube. The washes were spun down in a clinical centrifuge. The resin was suspended in 10 ml 2x NETS, for a final volume of ~13 ml.
To run a column, 1 mg total RNA was diluted to 1 ml with 10mM Tris-Cl pH 7.4, and then mixed with 1 ml resin as prepared above in a 2 ml Biorad minicolumn that is sealed on the bottom. The column was capped and the RNA was allowed to bind to the resin by placing on a rotator or nutator for 1 hour at RT. The bottom of the column was broken off after removing the cap, and mounted on a 15 ml conical tube. The contents were then allowed to flow through. After all the liquid had drained from the column, the column was removed to a new tube, and the resin was washed twice with 0.7 ml of 1x NETS. The column was removed to a new tube, and the resin washed three additional times with 0.7 ml of 1x NETS. To elute, the column was removed to a new 15 ml conical tube, and 0.7ml elution buffer (10mM Tris-Cl pH 7.4) that was preheated to 70°C was added twice in succession. To verify the enrichment, a gel was run of 5-7 ul of the contents of each of the conical tubes. Generally, the flowthrough and first two washes had very bright ribosomal RNA bands. Washes 3-5 generally had very little RNA. The eluate usually had much weaker rRNA bands and a brighter smear signifying mRNA. Usually, about 30-50% of the eluate was rRNA, representing about a 30-60 fold enrichment of mRNA.
The eluate was then divided into two 1.5ml eppendorf tubes, and extracted twice with CHCl3 to remove cellulose. The RNA was then precipitated with 0.3M NaOAc, and one volume of isopropanol at -20°C for 1 hour, then spun 30 minutes in microfuge at 4°C. The mRNA pellets were then washed with 75% EtOH, and spun for 15 min at 4°C. The pellets were allowed to air dry for about 10-15 minutes, then resuspended in 10-20 µl DEPC-treated ddH2O, depending on pellet size. A spectrophotometer reading was taken using 1 ul in a microcuvette. The mRNA was then stored at -80°C in aliquots of 5-10ug to avoid thawing and re-freezing.