Affymetrix expression arrays generally only require 100-300 ng input of total RNA.
Note: This procedure is not suitable for making Total RNA for miRNA analysis - the complete Invitrogen Trizol procedure is suitable for extracting RNA for miRNA labelling.
Pelletted cells (<5x108 cells) are dissolved in 500µL Trizol then 100µL of chloroform is added and the mixture is vortexed, left on ice for 15min. then centrifuged (6500 x g ) for 30min. at 4C.
The upper aqueous phase is retained, mixed with an equal volume of 70% ethanol (in DEPC H2O) then the RNA is further purified using a Qiagen RNeasy kit. Briefly, the RNA/ethanol mixture is applied to a RNeasy column then centrifuged at 6500 x g for 1min. The eluent is re-passed through the column which is then washed with 700µL of buffer RW1. The column is washed twice with 500µL of buffer RPE then residual buffer is removed by spinning the column at 6500 x g for 1min. The purified RNA is eluted into a clean tube with 30µL of DEPC treated water.
If required, RNA should be concentrated by precipitation.
Add 1/10 volume of 3M sodium acetate (pH=5.2) and 2 volumes of ethanol. The mixture is cooled to -80C for 30min then RNA is pelletted by spinning (6500 x g ) for 20min. at 4C. The pellet is washed by adding 500µL of ethanol and spinning at (6500 x g ) for 10min. The supernatant is decanted and the pellet is left to dry for 60min. at room temperature. The RNA is dissolved in DEPC treated water and the concentration is determined.
Gel Electrophoresis of RNA
Note: all apparatus must be thoroughly washed with 10% SDS and rinsed with DEPC water prior to preparation.
- The gel is prepared by mixing 4mL of 5X gel running Buffer (0.2M morpholinopropanesulfonic acid [MOPS pH=7], 50mM sodium acetate and 5mM EDTA pH=8) with 4mL of 37% formaldehyde solution and 12mL of 1.5% molten agarose (w:v). The gel is cast when the mix cools below 50C.
- The RNA sample (4.5 µL or up to 20µg) is mixed with 2µL of 5X gel running buffer, 3.5µL of 37% formaldehyde and 10µL of formamide then heated to 55C for 15 min..
- 2µL of RNA loading buffer (50% glycerol, 1mM EDTA, 0.4% bromophenol blue and 0.4% xylene cyanol) is mixed with the RNA sample, loaded into the gel then electrophorised using 1x gel running buffer as the electrolyte.
- The RNA is visualized using ethidium bromide then photographed after destaining.