Poly(U) sepharose

●●●●●　Poly(U) sepharose　●●●●●

Poly(U)-sepharose 4B can be used to isolate poly(A)-containing RNA from total cellular, polysomal, or other RNA preparations. Although this is not the method of choice for isolating mRNA for cDNA synthesis (because oligo(U) shed from the column is a powerful inhibitor of priming for cDNA synthesis), this method is sometimes used if the poly(A)-containing RNA is to be analysed by rate-zonal (sucrose) ultracentrifugation or, especially, scintillation spectrophotometry.
Strict RNase-free technique must be used during this procedure, since both the desired product and the matrix are RNase sensitive. All buffers should be treated 0.05% DEPC, all glassware should be autoclaved, and sterile technique should be maintained. All glassware should also be siliconized. If the matrix quits working, discard it: it can easily be destroyed by RNase activity, especially upon prolonged storage.

1M NaCl
5.844 grams per 100ml - adjust pH to 7.5

HSB

		final conc.
40.91 grams	NaCl	0.7M
1.901 grams	Na2EDTA	10mM
7.90 grams	Tris HCl	50mM
250ml	formamide	25%
liter

LSB (per liter)

		final conc.
29.22 grams	NaCl	0.5M
1.901 grams	Na4EDTA	10mM
7.90 grams	Tris HCl	50mM
500ml	formamide	50%
liter

HSB and LSB should be made up as follows: Dissolve the solids in 400ml ddH2O. Add enough ddH2O so that the volume will be about 950ml after adding the formamide. Slowly add the formamide, then cool to 4C. (If the solution becomes turbid, stir overnight to redissolve the precipitate.) Adjust to pH 7.8, then adjust to 1 liter with ddH2O. Store under nitrogen in an airtight amber bottle.

EB (per 500ml)

		final conc.
1.441 grams	H4EDTA	10mM
1.00 gram	N-lauryl sarcosine	0.2%
1.2177 grams	Na2HPO4	10mM PO4
0.701 grams	NaH2PO4	10mM PO4
450ml	formamide	90%
500ml

Elution buffer should be made up as follows: Dissolve the solids in 950ml 90% formamide by stirring overnight at 4C. Adjust to pH 7.8 at 4C with 10N KOH, and adjust to 500ml with 90% formamide. Store at 4C under nitrogen in an airtight amber bottle.

NOTES: HSB, LSB, EB, and any other solution should be DEPC treated by adding 0.5ml DEPC (diethylpyrocarbonate) per liter buffer, and stirring overnight at 4C. Do not use the buffer for a few days after DEPC treatment. Use good quality formamide (i.e.MCB); the higher the grade, the better. Formamide should be deionized before use by stirring for 2 hr at 4C with 8-10 grams per liter mixed bed ion exchange resin.

Perform all steps at 4C.

Preparing the column:

Add 0.25 grams poly(U)-sepharose 4B to 20ml 1M NaCl. Stir very gently with a glass rod. Allow the suspension to settle for 20 min.

Decant off the upper half of the NaCl sol'n. Add 10ml fresh 1M NaCl, and allow the sepharose to swell overnight at 4C.

Decant off the upper half of the NaCl sol'n, and pour this into a syringe-style column. Allow the slurry to settle for 20 min.

Pass 20ml 1M NaCl through the column. NEVER LET THE COLUMN RUN DRY!

Pass 20ml EB through the column, and store in EB at 4C.

EtOH precipitate 1-4 mg total RNA, pellet, and dry under vacuum, as described in step 10. Dissolve the RNA in 2ml HSB by gently stirring with a baby stir-bar.

Add 20 mg N-lauryl sarcosine and 20 mg Na2EDTA, and continue stirring until completely dissolved (about 1 hr). Dilute the sample with 10ml HSB.

Equilibrate the poly(U)-sepharose column with HSB by passing 20ml HSB through the column.

Lower the buffer level over the column to just above the bed surface, and gently add the sample (so that the bed is not disturbed). Allow the sample to pass through the column at a flow rate of 3-4 drops/min. Collect 1ml fractions throughout the column run.

When the sample level reaches the surface of the bed, gently add 1ml HSB. Allow this to pass into the column, still at 3-4 drops/min. Then pass 20ml HSB through the column at the gravity flow rate.

When the HSB wash level reaches the surface of the bed, add 1ml LSB. Allow this to pass into the column, then pass 10ml LSB through the column.

When the LSB wash level reaches the surface of the bed, slow the column to 4-5 drops/min., and add 1ml EB. Start collecting 0.5ml fractions. Keep adding 1ml aliquots of EB until 10ml EB have passed through the column. Store the fractions at -20C.

Pool the first 3ml of EB to pass through the column (after setting aside 100-150ul of each fraction for A260 analysis, step 9), and apply this to a 1x20cm G-50 sephadex (50-150) column equilibrated in ddH2O. Collect 100 1ml fractions.

Dilute 100ul of each fraction (from both the poly(U)-sepharose and G-50 sephadex steps) with 0.9ml ddH2O and determine the A260 spectrophotometrically (or count aliquots by scintillation spectrophotometry). The poly(A)-containing RNA should come out as a tall, sharp peak in fractions 12-20.

Pool the G-50 fractions containing the poly(A)-containing RNA, and precipitate by adding 1/10 volume of 3M NaOAc and 2.5 volumes of ice cold EtOH. Store overnight at -20C, centrifuge at 15KRPM at 4C in an SW27 rotor, and discard the supernatant. Dissolve the RNA pellet in ddH2O to a concentration of about 1ug/ul, and store at -70C.

J. Vaughn (personal communication)
Lindberg, Persson, and Philipson 1972 J. Virol. 10:909
Lindberg and Persson Meth. in Enz. pp497-499 (Nucleic acids,
Nucleotides, and Derivatives pp57-59)
Pharmacia Product Report; Poly(U)-sepharose 4B