Evaluation of Spermatozoal Rna Extraction Methods

Abstract

The study was aimed to compare different RNA isolation methods from sperm and testicular tissue. A suitable sperm purification technique is required as sperm RNA yield is strongly correlated with the number of sperms. Goat (Capra hircus) semen was purified by swim-up method. Motile sperm were selected for the process of RNA isolation. Four different RNA isolation methods namely cold Trizol, Gunidinium thiocyanate method and SDS lysis method and silica column based spin column method were used to isolate total RNA from sperm and testicular tissue. The hot Trizol method yielded good amount of RNA with less contamination of DNA and proteins. The modification of heating the sample in Trizol and an additional chloroform phase separation step increased the yield of total RNA. The modified method yielded highest amount of RNA for both tissue and sperm samples which was 139.5 ng/μL and 38.6 ng/μL respectively. The RNA integrity was high as was defined by a sharp 28S and 18S rRNA band in the agarose gel. The A260/A280 ratio was 2.1 and 1.923 for tissue and sperm samples which signifies purity of RNA. The mRNA was also reverse transcribed and GAPDH amplified. A modified Trizol method was developed which resulted in increased yield (38.60 ng/μL) and purity (A260/A280: 1.923).