Comparison of Methods to Extract Total RNA from Diverse Marina Sponges for the "Porifera Tree of Life" Project

Comparison of Methods to Extract Total RNA from Diverse Marina Sponges for the "Porifera Tree of Life" Project

According to phylogenetic and paleontological evidence, sponges and their fossils represent the earliest living branch of the multi-cellular animal evolution. Therefore, more in depth laboratory studies of sponges and their phylogeny can provide information on the origin of multi-cellular animals, survival techniques, and medicinal treatments. As part of the NSF-funded "Porifera Tree of Life Project, also known as PorToL (www.portol.org), researchers in the Lopez laboratory, have been generating molecular sequences from a select group of nuclear genes to refine the sponge phylogeny. My goals were to compare and analyze the best preservation methods for total RNA extraction. Total RNA from a variety of sponge samples, which had been preserved in either a) ethanol, b) -20 oc freezing, or c) Trizol reagent, was extracted. Subsequently, final yield and purity of each total RNA sample were recorded. Essentially, the purpose of these analyses was to develop a standardized and reliable protocol. Some of the species that were analyzed are part of the 127 target sponge families. Using data collected from more than twenty different sponge samples, Nanodrop purity readings ranged from 1.6 to 2.0 with a pure reading for RNA around 2.0. Additionally, it has been experimentally determined that total RNA extracted from sponges preserved in Trizol reagent were generally higher than sponges preserved in ethanol, with a range between 100 ng/gL to over 2,000 ng/gL. In conclusion, Trizol is a better preservation method for most sponge samples; however, Hexactinellid species generally yielded less total RNA probably due to low tissue mass.