Maximizing Total RNA Yield from TRIzol Reagent Protocol: A Feasibility Study

This work aimed to optimize RNA isolation from samples of limited size using TRIzol reagent.Investigated were the effects of certain variables, especially the amount of TRIzol reagent used in homogenization of samples and exposure to freezing conditions, on total RNA isolation from vertebrate tissue.Initially, it was possible to optimize the amount of TRIzol used for the initial homogenization of amputated zebrafish caudal fin tissue.Samples were then prepared in three groups; RNA was isolated immediately following homogenization (0 hour) and after 1 hour and 24 hours of being snap-frozen in a -80C freezer.Lastly, since extraction of RNA in the aqueous phase involves elution with 24:1 chloroform/ isoamyl alcohol after the initial homogenization step of the protocol, also examined was how the volume of chloroform/ isoamyl alcohol impacted yield.The experimental techniques were then applied to isolating RNA from regenerated zebrafish tissue 24 hours post-amputation of the caudal fin for a study investigating gene regulation in regenerated tissue.Functional studies included cDNA synthesis and sequential RT-PCR to evaluate quality mRNA extraction by employing these optimized methods.After additional validation, the techniques were then applied to extraction of total RNA from a single zebrafish embryo at 4-cell, high, and 24 hpf stages of development.It was observed that this optimization process yields quality RNA (RIN ~ 10.0), and there exists significance in the amount of reagents applied to a sample versus the amount of RNA obtained from the extractions.