Self Org

...molecular self organisation and evolution

  • Increase font size
  • Default font size
  • Decrease font size
Home Methods and Protocols
Methods-Protocols

Desalting RNA oligomers

E-mail Print PDF

Depending on the size of the RNA oligomers one of the following methods can be chosen:

1. HPLC with a Triethyl-amine-acetate (TEAA) buffer

Column: Reverse Phase Column (RP18 = C18)

Eluents:

A: A mixture of Triethyl-amine and acedic acid (100%) in 1:1 ratio, adjusted to pH 7.0 with trietylamine or acedic acid. Final concentration 0.1M

B: 25% Acetonitrile in eluent A.

 

2. Sephadex G-25 column

3. Ion exchange beads

4. Dialysis  (the right cut-off size must be chosen !!)

 

MALDI-TOF-MS-Analysis of RNA oligomers

E-mail Print PDF

A good MALDI-TOF-MS analysis of RNA and DNA oligomers can be quite tricky to perform. Here are some hints that help to do a successful measurement:

1. RNA/DNA preparation

The oligomers shall contain as little salts as possible, especially no alkaline (Na+/K+) and alkaline earth metals (Ca2+, Mg2+).

Ammonium (NH4+) is fine.

The following procedures can be used to desalt the oligomers (depending on the oligo size) (s. also RNA desalting protocols).

  • HPLC with a TEAAA buffer
  • Sephadex G-25 column.
  • ion exchange beads (e.g. Dowex 50WX8, 200-400 mesh, ammonium form) [1]
  • dialysis  (the right cut-off size must be chosen !!)

2. Matrics

(i) 2′,4′,6′-Trihydroxyacetophenone monohydrate (THAP) matrix (recommended for short RNA and DNA oligomers).

To a saturated solution of 2′,4′,6′-Trihydroxyacetophenone monohydrate (e.g. FLUKA91928 or FLUKA41711) in ethanol (or iso-propanol) (sonicate !) add a 0.1 M aqueous solution of ammonium citrate (NH4+)2-H-citrate (30% v/v). Keep the mixture at 4°C and keep it no longer than 2-4 weeks.

(ii) 3-Hydroxypicolinic acid (3-HPA) matrix

To a saturated aqueous solution of  3-Hydroxypicolinic acid (e.g. FLUKA56197) add a 0.1 M aqueous solution of ammonium citrate (NH4+)2-H-citrate (30% v/v).

3. MALDI-sample preparation

(1) Clean the sample tray carefully with acetone, ethanol, water, acetone.

(2) pipette 1-4µl of matrix on each spot (some trial and error is needed to choose the best matrix for a concrete analytical problem)

(3) add RNA sample to the matrix solution on the spot (the amount depends on the sensitivity of the instrument - 10E-12mol analyte is a good starting point)

(4) let the analyte and matrix dry together at room temperature (Vacuum can speed up drying process, e.g., tray in evacuated exsiccator)

(5) analyse the sample in the spectrometer

4. MALDI measurement

  • If you have a deflector machine, please try 'linear mode' for longer oligomers > 6mer
  • adjust parameter set to RNA/DNA oligomers - for longer oligomers you sometime need relative high laser power to co-evaporate the oligomers with the matrix
  • try to find a default oligomer method on your spectrometer and alter parameters based on this method.

 

Further reading and links:

____

[1] The conversion of the H+ form of Dowex 50w8 resin to the NH4+ can be achieved by using a 2-5% NH4Cl or NH4OH solution. Please choose according to the counterions you want to have on your column. Wash resin beads with water until the desired pH is reached.

The conversion of the H+ form resin to the NH4+ can be done using a 2-5% NH4Cl or NH4OH solution. You may want to choose based upon which one has less counterion impact on your process, the NH4Cl will throw off a HCl, while the NH4OH will throw off a H2O. Once converted you will want to wash till the eluate is an acceptable pH for your process.