How can I decrease ribosomal RNA (rRNA) contamination using Oligotex?
The standard Oligotex procedures provide significant enrichment of poly A+ RNA. However, for a somewhat higher enrichment of mRNA, the following enrichment steps can be performed:
- For Oligotex mRNA protocols: following step 5, add equal amounts of RNase-free water and Buffer OBB. (If following the Oligotex mRNA Protocol using Oligotex Direct mRNA Buffers, use 1 part Buffer OL1 plus 4 parts Buffer ODB). Mix the contents thoroughly by pipetting or flicking the tube. Repeat steps 3–5, and then continue the relevant protocol with step 6.
- For Oligotex Direct mRNA protocols: for applications where highly purified mRNA is necessary (e.g., cDNA library construction), the full protocol rather than the shortened protocol is recommended. To clean up a sample with too much rRNA, do another round of purification following the optional protocol in Appendix D of the Oligotex handbook 'Oligotex mRNA Protocol Using Oligotex Direct mRNA Buffers', or use one of the Oligotex mRNA Kits.
Additional general tips:
- Add 0.5% SDS or LDS to the buffers in hybridisation and wash steps
- Double the volume of binding buffers without changing the amount of Oligotex Suspension
- Carry out binding/hybridisation with 300-400 mM NaCl (instead of 500 mM)
- Wash with 100 mM NaCl (instead of 150 mM)
- Use 10 mM of EDTA in the binding buffer (instead of 1 mM)