chapter  28
11 Pages

Restriction Endonucleolytic Analysis of Differentially Expressed Sequences: READS

I. Introduction 229

II. Materials and Methods 230

A. Reagents 230

B. Enzymes 230

C. Oligonucleotides 230

1. T7-Sal-Oligo d(T)18-V 230 2. READS PCR Primers 230

3. Fly Adapters 231

III. Experimental Protocol 231

A. General Precautions 231

B. Reagents To Be Prepared in Advance 231

1. Annealed Fly Adapter 231

2. Labeled PCR Primer 232

3. Labeled Molecular Weight Marker DNA 232

C. cDNA Synthesis 232

D. Restriction Enzyme Digestion of the cDNA 233

E. Ligation of Digested cDNA with Fly Adapter 233

F. PCR Amplification 233

G. Sequencing Gel Analysis 234

H. Autoradiography 234

I. Recovery of cDNA Bands from the Gel and PCR Amplification 234

J. Processing of the PCR-Amplified Samples for Sequence Analysis 235

K. Data Documentation 235

L. Technical Comments 235

IV. Discussion 236

References 237

I. INTRODUCTION

Profiling of cDNA expression patterns has become a major tool of molecular biology and proved valuable in a range of problems from fundamental studies of biochemical mechanisms to the classification of human tumors.2-5 The largest number of studies has been performed by

hybridization of labeled cDNA to arrays of oligonucleotides or cDNA fragments6-8 and, with rare exceptions, the arrays represent only previously detected transcripts. Large-scale independent confirmation of these results has rarely been performed. In addition, very recent studies indicate that there may be much more transcriptional activity in cells than would be suggested from the known or predicted protein coding genes.9,10 Methods for gel-based display that are independent of prior knowledge about the transcripts have been described for expression analysis.11-17 These methods are applicable to any eukaryotic organism. Among the gel-based display approaches, READS− 1,14,15,18,19 (Restriction Endonucleolytic Analysis of Differentially Expressed Sequences) gel display process (as outlined in Figure 28.1) involves selective PCR amplification of the 3-end restriction fragments of the cDNAs based on the N1N2 nucleotides 5-to the poly-(A) tail of the message. As a result, this approach produces well-defined products from each polyadenylated transcript and, in general, produces only one major product per poly-(A) site on the transcript. It has a wide dynamic range and predicts changes in the relative levels of different RNA species. The approach does not require any specialized equipment and is suitable for use even in small laboratories. READS can be carried out even with limited amounts of total RNA and hence can be applied to small tissue biopsies. The present chapter provides a detailed protocol evolved over several years of experience that has given very consistent results in the authors’ laboratories.