Protocol

Maize Tissue Preparation and Extraction of RNA from Target Cells for Genotyping

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. Where the quantity of isolated material is limiting, amplification can be used to increase the amount of product. 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 the preparation of acetone-fixed and paraffin-embedded maize seedling tissue sections. Once the sections are prepared, the PALM MicroBeam System, with its Laser Microdissection and Pressure Catapulting (LMPC) technology, is used to cut out cells of interest and "catapult" isolated tissues into collection caps. This tissue can then be used for RNA extraction.