Comp. by: NNadaraja Date:28/5/07 Time:15:59:47 Stage:1st Revises File Path://spiina1001z/womat/production/PRODENV/0000000001/0000003791/ 0000000016/0000584924.3D Proof by: QC by: ProjectAcronym:bs:MIE Volume:25002 CORRECTED PROOF CHAPTER TWO Identification of Modified Residues in RNAs by Reverse Transcription-Based Methods Yuri Motorin, Se ´bastien Muller, Isabelle Behm-Ansmant, and Christiane Branlant Contents 1. Introduction 22 2. Reverse Transcription (RT)–Based Methods for Detection of Modified Residues 24 2.1. Reverse transcriptases used for RNA analysis 26 2.2. General protocol for primer labeling and reverse transcription of RNA 26 3. RNA Extraction from Various Cell Types 29 3.1. Protocol for the extraction of total RNA from archaeal or eukaryal cells 29 3.2. Protocol for the extraction of total RNA from yeast cells 31 4. RNA Modifications Detectable After Specific Chemical Treatment 32 4.1. Detection of inosine using glyoxal treatment followed by RNAse T1 hydrolysis 32 4.2. Detection of pseudouridine (C) residues 34 4.3. Protocol for the detection of pseudouridine residues in RNA using CMCT modification 36 4.4. Complementary approaches for C detection 41 4.5. Detection of 5-methylcytosine (m 5 C) 41 4.6. Detection of 7-methylguanine (m 7 G) 42 4.7. Detection of 2 0 -O-methylated nucleotides using OH  cleavage or 2 0 -OH reactivity 42 4.8. Protocol for detection of 2 0 -O-methylated residues in RNA using OH  cleavage 43 4.9. Detection of 2 0 -O-methylated nucleotides by use of low dNTP concentrations 44 Methods in Enzymology, Volume 425 # 2007 Elsevier Inc. ISSN 0076-6879/07, DOI: 10.1016/S0076-6879(07)25002-5 All rights reserved. Laboratoire de Maturation des ARN et Enzymologie Mole ´culaire, Nancy Universite ´, Faculte ´ des Sciences et Techniques,Vandoeuvre-les-Nancy Cedex, France 21