April 12, 1998
Oligo dT isolation is a very useful method for isolating sequenceswith a poly A tag. Normally people use this column on whole cellpreparations of mRNA but in this lab it is mainly used to isolatesynthetic RNA molecules that have a poly A portion. The celluloseresin has oligo-dT covalently attached to it which form specificWatson-Crick base pairs with the target oligo-A tract. Those oligonucleotidesthat do not have complementary sequences will not bind to theresin and therefore flow through the column. Generally, a washstep is included before elution to lower the salt concentrationand reduce nonspecific binding. Elution can proceed with wateror with mild base. There are different resins available but Oligo-dTtype7 from Pharmacia seemed to be very robust. Oligo-dT agarosecan be made with biotinated oligo-dT and strepavidin agarose butthis seems not to work as well as the cellulose. The binding capacityseems to be much less than the cellulose based resin for whateverreason. The cellulose resins tend to have a very slow flow rate,but the detergent in the binding buffer seems to help. Also, theresin is very fine and some of it tends to flow through the column.A quick spin in a 0.2um eppendorf filter will easily remove theresin.
1M NaCl, 20mM Tris pH 8.0, 1mM EDTA, 0.2% Triton X-100
Note: other nonionic detergents such as Tween-20, NP-40, or CHAPSmay be used. The protocol seems to be very sensitive to the amountof each detergent. Experience from others in the lab suggest that0.2% Triton X-100 works best.
0.2% Triton X-100
|Triton X-100 (0.1%)|
Data kindly provided by Tim Heuer based on purification of RNA-peptidefusions.
Also, if you are purifying fusions, then you may want to 1mM DTTand 10mM EDTA to chelate all of the magnesium and dissociate boundribosomes. Some people like to add BSA ~50ug/ml to get rid ofnonspecific interaction but this is optional.
0.3M NaCl, 20mM Tris PH 8.0, 0.1% Tween-20 (the duplex is stablefor 0.3M salt, you may want to go lower if purity is a major factor).Detergent may be omitted in this step if so desired.
Note: others have gone down to as little as 100 mM without losinga considerable amount of material.
Water, or 15mM NaOH, with or without detergent. Add 1mM DTT forprotein selections. Alternatively, a competing oligo may be usedfor elution.
If using a competing oligo, the oligo should have a poly-T tractthat is completely complementary to the poly-A tract in your RNA.You should also have a sequence complementary to the RNA segementupstream of the poly-A tract to ensure that the competition willbe efficient.
Note: Water is very robust in eluting bound molecules. However,this may not be optimal for the elution of protein-RNA fusions.If performing thiopropyl-sepharose, having detergent helps thebinding step. Elution is more efficient with less detergent inthe buffer. Also, buffers such as Tris or Hepes and EDTA can beincluded.
1) Weigh out the appropriate amount of oligo dT - cellulose. Ifusing a disposable Bio-Rad column setup, no less than 20 mg canbe used. Typically, the binding capacity for the Pharmacia type7brand is around 200 nanomoles/gram of resin.
2) Wash the resin twice with ddH20.
Optional: Wash the resin thoroughly with 0.1N NaOH and incubatefor 15 min. Then wash with water repeatedly until pH ~7.
3) Wash resin 2 times with the binding buffer and resuspend inthe desired volume with binding buffer.
Optional: Add non-specific RNA or DNA (~0.5 ug/ml of resin) toreduce background binding.
4) Add sample to the resin in the appropriate binding buffer (ifsmall scale, this can be done in an eppenfdorf tube). Incubateat 4 ° for ~ 30 minutes while rotating.
5) Pour into a Bio-Rad disposable column and collect flow through.(You can discard this or save for analytical purposes). Alternatively,you may wish to reload the flowthrough through the column to getall those last remaining molecules.
6) Wash with 10 column volumes of binding buffer.
7) Wash with 10 column volumes of wash buffer.
8) Elute with 2 column volumes of elution buffer (you may wantto explore just how much elution your particular molecule needswith radiolabled molecules).
9) Remove resin from you eluate by filtering through a 0.2um spinfilter.
Note: if using the oligo elution. Plug column and add 500 ul ofWash Buffer as well as having a 100-fold molar excess of the competingoligo over RNA template. Incubate column for 1h at 37 °C whilerotating. Then drain 500ul which should contain your eluted RNAbound with the competing oligo. This procedure is primarly usefulfor eluting RNA-protein fusions under gentle, nondenaturing conditions.
You may wish to use your oligo-dT resin again, although it isfairly cheap. If you are one of those penny pinchers then followthis protocol:
Retrieve resin from column and wash with 0.1N NaOH for ~15min.Wash thoroughly with water and resuspend in TE. Keep at 4 °until use again.
I quantitated how much labeled protein-RNA fusion molecules Icould purify by oligo dT cellulose. I started with 20ul of myin-vitro translation reaction which contained about 80 pmol ofRNA that had a poly-A tag. If we assume nearly 2% of the RNA wasfused to the protein then around 1.6 pmols of RNA was fused. Theprotein was labeled with 35S and I quantitated the amount of 35Scounts before the oligo dT isolation and after the isolation.I do not have the numbers for how much stuck to the column andhow much eluted, but I retrieved 80% of my starting material asquantitated by Phosphorimager. Conferring with others who haverun this protocol, between 80-90% recovery seems reasonable.
Maniatis "Guide to Molecular Cloning"
Current Protocols in Molecular Biology (The Red Book)