purification of in vitro transcribed RNA

For this you will need to run you transcription product on a denaturing acrylamide gel:

N.B.: Urea takes time to dissolve and may require gentle warming (40-50°C) so try to prepare the two urea containing solutions in advance.

solutions to prepare

• 10% w/v ammonium persulfate in water (can be stored at 4°C)
  

• 100 mL 20% acrylamide/urea solution:

49.85 g UREA

0.5 g N,N methylene bisacrylamide

50 mL acrylamide 40% stock (Bio-Rad 161-0140)

10 mL TBE 10X

• TBE 10X :
  

108 g Tris base

56 g boric acid

40 mL 0.5 M EDTA pH8.0

double distilled H2O (ddH2O) to 1L

• 100 mL of TBE/UREA solution:
  

dissolve 49.85 g UREA in 80 mL autoclaved H2O

add 10 mL 10X TBE

top up to 100 mL with autoclaved H2O

• formamide loading buffer:

10 mL formamide

10 mg xylene cyanol FF

10 mg bromophenol blue

200 µL 0.5 M EDTA pH8.0

Casting and pouring the gel

Choose the adapted % for your transcript :

dye migration according to denaturing acrylamide %

Then cast a 1 mm x 20 cm x 20 cm gel, taking care to clean both glass plates an

d silanizing one of the two plates (silanizing liquid available from Lonza).

• gel recipe:
  

X mL acrylamide/UREA8.3M

TBE/Urea to 40 mL final

240 µL 10% ammonium persulfate

50 µL TEMED

This gel polymerizes very fast due to the presence of urea, be careful !!!!!

Avoid leakage by pouring the gel as horizontally as possible, bubble formation should be avoided but if bubbles appear, you can always try to get rid of them by playing with the left/right inclination of the assembled glass mold.

clean the wells with a syringe equipped with a bent needle.

pre-run the gel in TBE 1 X for 30-60 min at 500V

Sample preparation

During the pre-run, add 40 µL of loading buffer to the 50 µL transcription product redissolved in EDTA and heat 5 minutes at 95°C,

chilled on ice,

stop the pre-run f the gel,

clean the wells a second time, with a syringe equipped with a bent needle prior to load,

load the samples carefully and run the gel keeping the same conditions as in the pre-run.

Monitor RNA

RNA should be accurately visualized by UV shadowing : using a UV lamp (I used 254 nm to better see the RNA, but higher lambdas are considered less damaging for RNA) and a silica coated plastic sheet (usually used in Thin layer chromatography).

Uncast the gel when the run is over and dispose it on a cellophane film, put the silica sheet under this cellophane and directly expose the gel to the UV source, which gives this from top to bottom:

UV light

gel

cellophane

silica

Alternately, if the RNA was transcribed with radioactive alpha 32P UTP, you just have to monitor its presence by autoradiography.

Cut the band of interest, chop it in very small slices.

Elute it in elution buffer:

elution buffer can vary depending on the type of experiment that follows the purification.

If you want to 5'end label the RNA for instance it is advised not to use tRNA in the elution buffer, whereas this is not a problem for RNA that has been body labelled with radioactive UTP during transcription.

For 5' end labeling it is also important NOT TO USE DEPC anywhere since this compound may inhibit dephosphorylation and phosphorylation steps.

elution buffer with tRNA (400 µL should be sufficient) :

0.75M ammonium acetate

0.1% SDS

10 mM MgOAcetate

0.1 mM EDTA pH8.0

add at the last minute

25 ng/µL bovine or E.coli tRNA

10 µL/mL phenol buffered with 0.1M citrate pH 4.3

elution buffer without tRNA (1 mL should be sufficient):

50 mM Tris-HCl pH7.5

300 mM NaOAcetate

0.5% SDS

Incubation can vary but minimum is of 2 hrs at room temperature (tried with no tRNA) or 37°C (tried with elution buffer with tRNA and phenol) under vigorous shaking.

Once elution is performed, treat with acidic phenol, chloroform and precipitate overnight with NaOAc and pre-chilled absolute ethanol.

Avoid using ammonium acetate if the RNA is prepared to be used for radioactive 5' end labeling.

resuspend RNA in autoclaved water.