RNA Sequencing and
rRNA 2'-O-Methyl Mapping Protocol

We have sucessfully used this protocol to visualize more than 90% of the 55 2'-O-ribose methyl sites in S. cerevisiae ribosomal RNA.

To see what kind of results to expect, see the rRNA primer extension gels for the yeast methylation guide snoRNAs (click on "gel" in the "Functional Verification" column).

Todd Lowe (02/99)

  1. Labelling mapping primers

    2. rRNA mapping primers I use are generally 22-26 nt long. Design primers so that target 2'-O-meths are 10-80 nt upstream of primer annealing site.

    Mapping primers should be PAGE purified before use. This is important unless you get very clean oligo synth (we get ours from Gibco BRL, and they definitely need to be purified). If you don't PAGE-purify your oligos, you may get a non-specific "shadow" on all your gels (from 0-30 nt above your primers) when running out primer extension products. When page purifying, I generally recover 20-30% of what I load on the gel. Quantitate and adjust purified primers to 12.5 uM. Make sure to use all RNAse free solutions.

    32P End-labelling primers:

    In 50ul rxn
===========
 2 ul PAGE purifed primer (12.5 uM) (25 pmol total)
 5 ul 10x PNK buffer (I use NEB or Promega)
 2 ul T4 polynucleotide kinase (10 U/ul) (NEB or Promega)
10 ul 32P gamma-ATP (10mCi/ml, 3000 Ci/mmol spec. activity), no more
       than 2 weeks after ref date to get strong bands
31 ul ddH2O

Incubate 37C for 30 min, 95C for 3 min, chill on ice

  2. Spin column purify labelled oligos

    3. This step is optional, but you will get cleaner looking gels and the gel running buffer won't be so hot. You can use pre-made push columns or spin columns, I make my own:

    (From Maniatis)

    In my experience, if the primer conc. is too low by 1/2 or more (i.e. your columns gave too much run through b/c they weren't equilibrated with washes properly), the 2'-O-methyl band intensity for primer extensions goes way down.

  3. RNA sequencing rxns (for size ladder next to 2'-O-meth primer ext rxns)

    4. (Buffer recipes below) 

    Anneal:

 1 ul total RNA (2 ug/ul)
 1 ul 10x RT-Mg buffer (Read: RT minus Mg, NO Mg in this buffer!)
 6 ul labelled, PAGE-purifed mapping primer (from above, 
       0.25 pmol/ul, 1.5 pmol total)
 2 ul ddH2O
=====
10 ul total

Anneal @ 55-60C for 4 min, chill on ice immediately

Primer Extension:

In each of 4 tubes (A,C,G,T)

 1 ul 5x dNTP mix
 1 ul 5x ddNTP mix (one tube for each ddA, ddC, ddG, OR ddT)
 1 ul RVT mix (1 U AMV-RT/ul, 1X RT+Mg buffer, 24 mM MgCl2)
 2 ul annealing mix (from above)
=====
 5 ul

  4. dNTP concentration-dependent 2'-O-methyl assay

    5. Anneal:

 2 ul total RNA (2 ug/ul)
 1 ul 10X RT-Mg buffer
 6 ul labelled, PAGE-purified primer (~0.25 pmol/ul)
 1 ul ddH2O
=====
10 ul

Anneal @55-60C for 4 min, chill on ice immed

Primer Extensions (one tube for each dNTP conc)

 1 ul RVT mix (1 U/ul AMV RT, 1x RT-Mg buffer)
 1 ul 25 mM MgCl2
 1 ul 5x dNTP mix (5 mM [Normal] or 0.02 mM dNTP [low])
 2 ul anneal mix (from above)
=====
 5 ul

Notes

Buffers

- all soln's DEPC-treated / RNAse free 
10X RT+Mg buffer (store @ 4C)
-----------------------------
500 mM Tris-Cl pH 8.6 @ 25C
600 mM NaCl
 60 mM MgCl2
100 mM DTT

10X RT-Mg buffer (store @ 4C)
-----------------------------
500 mM Tris-Cl pH 8.6 @ 25C
600 mM NaCl
100 mM DTT

5X dNTP solution for RNA sequencing (store -20C)
-----------------------------------
  3 ul 100 mM dATP
  3 ul 100 mM dCTP
  3 ul 100 mM dGTP
  3 ul 100 mM dTTP
 18 ul 10X RT+Mg buffer
150 ul dd H2O

5X ddNTP solutions (store -20C)
------------------
20 ul 5 mM ddNTP (ddA, ddC, ddG, OR ddT)
10 ul 10X RT+Mg buffer
70 ul dd H2O

Stop Soln / Loading Dye mix (in case you want to mix your own)
===============
 22.4 ul 2.5 % bromophenol blue
 22.4 ul 2.5 % xylene cyanol
 56.0 ul 10X TBE buffer
  1.3 ml formamide

Credits: