Protocol

T7-Based RNA Amplification for Genotyping from Maize Shoot Apical Meristem

¹Department of Agronomy, Iowa State University, Ames, IA 50011, USA
²Department of Genetics, Development, and Cell Biology; and Center for Plant Genomics; Iowa State University, Ames, IA 50011, USA

³Corresponding author (schnable{at}iastate.edu)

INTRODUCTION

The use of RNA for genotyping analysis can be advantageous because transcriptomes are significantly smaller than genomes and typically contain far fewer repetitive sequences. Laser capture microdissection (LCM) has been used successfully to isolate sequences (especially rare transcripts) that accumulate in specific tissues. The amount of RNA collected in a standard microdissection is often insufficient for global gene expression analysis but can be increased via linear amplification. Upon conversion to cDNA, the product serves as template for 454 sequencing to produce expressed sequence tags for subsequent SNP analysis and detection. This protocol describes how to amplify RNA extracted from laser-dissected and captured tissues and cells.