Protocols

Protocols are available for extracting total RNA for both spotted and Affymetrix arrays. The Centre has adopted robust protocols that we recommend to be used with our spotted microarray slides.

Affymetrix Genearrays require only 100-300 ng input of total RNA, whereas spotted cDNA arrays require a minimum of 20 ug.

Note: This procedure is not suitable for making Total RNA for miRNA analysis - the complete Invitrogen Trizol procedure should be used for extracting RNA for miRNA labelling.

RNA Extraction

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.

RNA Precipitation

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.

cDNA Synthesis & Dye Coupling

Total RNA (>20µg) is dissolved in 20µL of DEPC H2O. 2µL of anchored polyT(V)N (2µg/µL) is added and the mixture is incubated at 70C for 10min. The sample is then placed on ice.
The sample is mixed with 6µL of 5X Superscript II buffer, 2µL of 0.1M DTT, 2µL of Superscript II (200U/µL) and 0.6µL of aminoallyl (aa) dNTP mix (25mM dATP, 25mM dGTP, 25mM dCTP, 10mM dTTP and 15mM aa dUTP) then incubated at 42C for 2.5 hours.
The RNA is hydrolysed by adding 10µL of 0.25M NaOH, 10µL of 0.5M EDTA (pH=8) and incubating the mix at 65C for 15min.. The reaction is neutralised by adding 15µL of 0.2M acetic acid.
The cDNA is purified using a QIAquick PCR purification kit. Briefly, the cDNA is mixed with 300µL of buffer PB then applied to the Qiagen column and centrifuged at 6500 x g for 1min.. The eluent is re-passed through the column. The column is washed twice with 600µL of buffer PE then residual buffer is removed by spinning the column at 6500 x g for 1min.. The sample is eluted into a clean tube with 90µL of Milli Q water.
The purified cDNA is dried under reduced pressure then dissolved in 9µL of 0.1M NaHCO3 (pH 9.0). The mixture is added to a Cy dye aliquot*, mixed then left to incubate at room temperature for 60min. in the dark.
The labeled cDNA is mixed with 41µL of Milli Q water then purified using a QIAquick PCR purification kit. Briefly, the cDNA is mixed with 250µL of buffer PB then applied to the Qiagen column and centrifuged at 6500 x g for 1min.. The eluent is re-passed through the column. The column is then washed twice with 600µL of buffer PE then residual buffer is removed by spinning the column at 6500 x g for 1min.. The sample is eluted into a clean tube with 90µL of Milli Q water. The purified labeled cDNA samples appear purple after being dried under reduced pressure.

*One Cy dye sachet (Amersham cat#PA23001 and PA25001) is dissolved in 73µL of anhydrous DMSO then 4.5µL aliquots are distributed into sealable tubes. Each aliquot is dried under reduced pressure, sealed then stored in a dessicator at –20C.

Hybridisation & Washing

We recommend the procedures outlined in the following Acrobat PDF file for hybridisation and washing of Oligonucleotide and cDNA microrrays.

An Acrobat PDF version of these protocols is available for printing.