Primer extension protocol for highly structured RNAs

Oligonucleotide purification:

Dissolve deprotected oligo in 200 µl of DNA sequencing stop-buffer. Load in 20-30 mm wide slot of 1.5 mm/20 cm 15-20% acrylamide/ 8M urea gel, and run until bromphenol blue dye reaches the bottom. Cut out the band under UV shadowing, put in 15 ml tube, and crush the acrylamide.

-Add 5 ml of SEB (0.5 M NaCl, 100 mM Tris, pH 8.1, 5 mM EDTA)

Shake overnight at 37 C, pellet down the acrylamide, wash the pellet with 2 ml SEB, pool upper phases, and filter through 0.45 um cellulose acetate filter.

C18-purification:

• attach C18 Sep Pak cartridge to 10 ml syringe
• wash with 10 ml acetonitrile
• wash with 15 ml of water
• apply sample in SEB to the column
• wash with 10 ml of water
• elute with 2.5 –3 ml 30% (v/v) acetonitrile in water, collect in a glass tube
• evaporate to dryness
• resuspend in 350 µl of water, extract with phenol/chl, add 50 µl 3 M NaoAc, pH 5.0, ethanol-precipitate. Measure OD and adjust concentration to 50 mM. Make an aliquot of 10 mM.

With this protocol even low amounts of oligo can be recovered.

Alternative purification:

Use 0.5 M (NH4)oAc, 0.1 SDS, 1 mM EDTA instead SEB

After elution (6-7 ml), extract with 5 ml of phenol/chloroform/isoamyl alchohol, concentrate by 2-3 extractions with isoamyl alcohol to 1 ml, and precipitate with 3 vol ethanol overnight at –80 C. Re-dissolve in 300 µl, extract with phenol/chl/iso, add NaoAc to 0.3 M, precipitate with 3 vol ethanol.

This protocol is recommended if the amount of oligo is high (if it has a very thick band under UV-shadowing).

Oligo labeling

Mix:

4.3 µl water

0.2 µl oligo (2 pmol)

2 µl of 5x buffer

3 µl gamma-³²P-ATP

0.5 µl kinase (5 U)

Incubate 30 min at 37 C, add 0.2 µl 0.5 M EDTA, 10 µl water, and heat 5 min at 65 C.

Keep frozen or use immediately.

Primer extension

Solutions:

0.4 M PIPES, pH 6.4

formamide with 2 mM EDTA

3 M NaCl

-co-precipitate RNA with a primer (0.1-0.2 pmol) in 0.5 ml tube.

Amount of RNA should be titrated, 10-50 mg total RNA is a reasonable range.

• wash 2x with 200 ul of 70% ethanol at RT
• dry on the bench, avoid overdrying
• resuspend in 3 µl of water
• add 3 µl of 0.4 M PIPES
• add 20 µl of formamide
• heat at 80 C for 3 min
• add 4 µlof 3 M NaCl (quickly), place at 65 C, and let cool down to 37 C overnight

(can be done in PCR machine or in 2 L of water heated to 65oC and left at room temperature)

• add 170 µl of water and 400 µl of ethanol, incubate on ice for 15 min, spin for 10 min at 14,000 RPM
• wash 2x with 200 µl of 70% ethanol
• resuspend in 23 µl of 1x RT buffer with 1 mM dNTP
• add 2 µl of RT (20 U), incubate 30 min at 37 C and 30 min at 50 C
• add 1 µl of 0.5 M EDTA, 5 ng of RNAse A per reaction, incubate at 37 C for 30 min
• add 150 µl of TE with 100 mM NaCl
• extract with phenol/chl, add 500 µl ethanol, incubate on ice for 15 min, spin down

dissolve in 50% formamide, analyse on sequencing gel

Contributed by Ruslan Aphasizhev.