RNA Detection

3.4 Purification of
18S and 25S rRNA
from Yeast Total RNA

The protocol for RNA isolation is derived from [13]. The aim of

this technique is to avoid any UV irradiation together with ribonu-

clease contamination that causes severe fragmentation of RNA

entrapped in agarose gels.

1. Prepare 1% low-melting agarose by mixing 1 g of agarose with
   100 mL of 1TBE and heating the preparation in a microwave
   oven at intermediate power. This percentage of agarose gel is
   appropriate for separation of 18S from 25S rRNA (Fig.1).

Fig. 1General overview of 2^0 - O-methylation quantification using RNP reconstruction for 18S and 25S rRNA.
Yeast 18S and 25S full-length rDNA were PCR amplified from plasmid pHW18 using specific forward
(containing a T7 RNA polymerase promoter) and reverse primers. The PCR templates were then used for
in vitro transcription and RNP reconstruction. In vivo 18S and 25S rRNA were gel-purified using a yeast total
RNA preparation and used for RNP reconstruction. In vitro rRNA and in vivo rRNA were equimolar mixed and
compared to yeast total RNA preparation and further used in different ratios for RiboMethSeq Protocol

Quantification of 2^0 - O-Me by RiboMethSeq 37