Title: Comparison of methods of extracting messenger Ribonucleic Acid from ejaculated Porcine (Sus Scrofa) Spermatozoa Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 21, 2013
Publication Date: April 1, 2013
Citation: Frankel, T.E., Theisen, D.D., Guthrie, H.D., Welch, G.R., Woods L.C. III. 2013. The effect of freezing rate on the quality of striped bass sperm. Theriogenology. 79(6):940-945. DOI:10.1016/j.theriogenology.2013.01.099. Technical Abstract: H. D. Guthrie, G.R. Welch, and L. A. Blomberg. Comparison of Methods of Extracting Messenger Ribonucleic Acid from Ejaculated Porcine (Sus Scrofa) Spermatozoa. Biotechnology and Germplasm Laboratory, Agricultural Research Service U. S. Department of Agriculture, Beltsville, MD 20705 The purpose of this study was to develop a method of isolating RNA from specific numbers of boar sperm to obtain repeatable recovery of RNA for transcriptome analysis. Comparison was made of different RNA extraction reagents (Qiazol and Trizol), different RNA isolation procedures after extraction and chloroform partition (RNeasy mini-columns and isopropanol precipitation), and different methods of quantitation (Agilent 2100 Bioanalyzer and NanoDrop 1000 spectrophotometer) using porcine testis tissue and sperm. The testis RNA exhibited 18S (2000 nt) and 28S (4000 nt) bands using the Qiazol-RNeasy and Trizol-RNeasy procedures with RNA Integrity Number values of 7.5-8.2. For sperm the 18S and 28S ribosomal bands were absent or reduced and RNA species < 2000 nt were most abundant using Qiazol and Trizol extraction reagents and RNeasy mini-columns. In contrast to the RNeasy column procedure, the isopropanol precipitation procedure for sperm showed a single, strong RNA band at about 25 and 170 nt for Qiazol and Trizol extraction, respectively. Compared to determination of sperm RNA concentration by the Bioanalyzer, the amount of sperm RNA calculated from absorbance at 260 nm using the NanoDrop was inflated by many-fold because of the high absorbance at 230 and 270 nm that was likely caused by the effects of guanidinium salts contained in Qiazol and Trizol that were not removed by the RNeasy column procedure or diluted prior to spectrophometry. By contrast, for testis RNA analyzed at a 1000-fold dilution there was no difference in RNA concentration determined by the NanoDrop and Bioanalyzer. The estimate of RNA concentration (ng/µl) in snap-frozen sperm analyzed on one Pico chip with the Bioanalyzer was linear from 1.4 to 14.5 ng/µl with a slope = 9.74 and intercept = -0.46 (r = 0.982, P < 0.05) over the range 0.2 to 1.6 billion sperm. The mean PCR primer efficiency of eleven pairs of primer sets for the genes tested was 1.008 in testis RNA with no false positives in the presence of non-reversed transcribed RNA. The purity of the ejaculates was investigated. A discontinuous Percoll sperm isolation procedure was tested, but could not be used because the presence of Percoll caused a 10-fold reduction in RNA concentration that was associated with a precipitate formed during the chloroform partition step. As a marker for proliferating germ cells, c-kit was undetectable by RT-PCR. Transcripts for E-cadherin and CD45 considered specific for epithelial cells and leukocytes, respectively, averaged 4 orders of magnitude less (0.01%) than the abundant transcript for protamine-1. Somatic cell contamination was also analyzed by flow cytometry to count the number of diploid cells in 100,000 events after propidium iodide staining of Triton-X-100 permeabilized semen samples containing 20 x 106 sperm cells. Ejaculates from different males contained 0.008% to 0.125% somatic cell contamination. Sperm RNA recovery appears to be equivalent for snap-freezing at -80oC with or without the use RNAlater and for non-frozen storage at 17oC for 24 h with RNALater. The methodology judged to provide the most representative porcine sperm RNA samples for subsequent transcriptome analysis was snap-freezing of 1-1.5 billion sperm at -80oC shortly after collection, isolation using the RNeasy Lipid Tissue Mini kit (QIAGEN) procedure, and quantification and quality evaluation using the Agilent 2100 Bioanalyzer.