Extraction of RNA from Small Amounts of Amphioxus Embryos for cDNA Synthesis by RT-PCR

INTRODUCTION

This protocol describes a rapid method for obtaining total RNA from several hundred (or fewer) amphioxus embryos. This protocol uses a commercially available spin column kit and is especially useful for experiments involving very small amounts of starting material, such as embryos injected with transgenes or morpholino antisense oligonucleotides. After the target embryos are sorted and rinsed, they are lysed and homogenized in lysis buffer prepared with guanidine isothiocyanate and containing β-mercaptoethanol to release RNA and inactivate RNases. Ethanol is added to the sample to promote selective binding of RNA to the silica-based membrane in the spin column. After applying the sample to the spin column to bind RNA, DNA contamination is removed by DNase treatment on the spin column. Finally, DNase and digested DNA are washed away, and high-quality RNA is eluted in RNase-free water. The resulting RNA can be used directly to generate cDNA by reverse transcriptase polymerase chain reaction (RT-PCR) for more sensitive applications such as quantitative PCR (Q-PCR).