The protocol below utilizes the Amersham Pharmacia mRNA Purification Kit (Cat. 27-9258-01) to purify mRNA from Total RNA.

Prepare Spin Columns (takes about 45 min)

• invert column several times to resuspend resin
• Remove Cap, place in 15 ml centrifuge tube, allow to drain under gravity
• Add 1 ml High Salt Buffer, allow to drain under gravity
• Add 1 ml High Salt Buffer, allow to drain under gravity
• Remove cloumn, discard buffer, replace column and apply RNA

Prepare RNA for Column

• Heat to 65 ° C, 5 min.
• Place on ice
• add 200 µl sample buffer

• place elution buffer at 65 ° C to pre-warm
• add RNA to Column, allow to soak in under gravity
• Spin Column (350g) 1237 RPM, 2 min.
• add 250 µl High Salt Buffer, spin 1237 RPM, 2 min.
• Repeat
• add 250 µl Low Salt Buffer, spin 1237 RPM, 2 min.
• Repeat twice
• Remove Column, discard buffer, place 1.5 ml tube inside centrifuge tube
• Replace column with its end inside the 1.5 ml tube
• add 250 µl Elution Buffer at 65° C
• Spin Column 1237 RPM, 2 min.
• Repeat elution 3 more times

Notes
For ethanol precipitation in a single eppendorf tube (I elute into screw cap tubes) spinvac RNA for 40 minutes to bring volume below 400 µl and then Ethanol Precipitate. Alternatively, elute into 2 ml screw cap tubes and use an equal volume of isopropanol for precipitation in place of ethanol).

Ethanol Precipitation:

add 1/10th volume 3M NaOAc add 10-20 µg linear acrylamide as carrier add 2.5 volumes 100% ethanol

Store the precipitate at -20 °C indefinately. To pellet the RNA spin at 13000g for 10-15 minutes.

The oligo-dT columns tend to leave a small amount of resin fines with the polyA RNA. Once the RNA is resuspended, the fines can be pelleted by a brief spin.

RNA is sensitive to degradation by high pH. ( I often use 1/2x TE to resuspend RNA, or 5 mM Tris pH 7.5)

RNA is very sensitive to contamination and degradation by nucleases. Always wear gloves when dealing with RNA. Keep RNA on ice as much as possible. Be very careful with all reagents that you use for RNA work. A primary source of conatimination is inadvertant touching of the lips of tubes. Most plastics are RNAse free. Dust is not RNAse free so be wary of leaving racks of pipet tips open to collect dust.

The recommended RCF for the columns is 350g. You can calculate the RPM according to the formula: RCF = 350 = 1.12r(RPM/1000)^2 or use this RCF calculator.