The Preparation of Human Spermatozoal RNA for Clinical Analysis

The recent identification of RNA as a component of mature spermatozoa necessitated the development of a reliable isolation protocol capable of yielding a high-quality substrate. In addition to the inherent difficulties associated with isolating RNA, the procedure as applied to sperm must overcome the resilient nature and reduced RNA content found within this cell type. Further, the protocol must be suited to the clinical setting. A reliable RNA isolation procedure optimized for this unique cell type is described. Ejaculate is collected, contaminating somatic cells lysed then spermatozoal RNA released by homogenization in a chaotrope. RNA is then purified from the homogenate by chromatography using a commercially available resin. The quality of isolated samples is assessed by PCR and RT-PCR. Once purity is established samples are suitable for numerous applications including amplification and probe synthesis. The reliable and consistent isolation of high-quality RNA from mature spermatozoa will aid in the development of new tools for the clinical assessment of male-factor fertility.

[1]  Angshumoy Roy,et al.  Non-invasive genetic diagnosis of male infertility using spermatozoal RNA: KLHL10 mutations in oligozoospermic patients impair homodimerization. , 2006, Human molecular genetics.

[2]  G. Ostermeier,et al.  Towards a better understanding of RNA carriage by ejaculate spermatozoa. , 2006, Human reproduction update.

[3]  R. Rowland,et al.  Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen. , 2006, Journal of virological methods.

[4]  M. Deng,et al.  Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR , 2005, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[5]  S. Krawetz.  Paternal contribution: new insights and future challenges , 2005, Nature Reviews Genetics.

[6]  G. Ostermeier,et al.  Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility. , 2005, Fertility and Sterility.

[7]  S. Krawetz,et al.  RNA in human sperm. , 2005, Asian journal of andrology.

[8]  G. Ostermeier,et al.  The controversy, potential and roles of spermatozoal RNA. , 2005, Trends in molecular medicine.

[9]  S. Krawetz.  [On the significance of RNA in human sperm]. , 2005, Zhonghua nan ke xue = National journal of andrology.

[10]  Zuo-min Zhou,et al.  A spermatogenesis-related gene expression profile in human spermatozoa and its potential clinical applications , 2004, Journal of Molecular Medicine.

[11]  G. Ostermeier,et al.  Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs , 2003, Biotechnology Letters.

[12]  J. Siffroi,et al.  Transcription in haploid male germ cells. , 2004, International review of cytology.

[13]  G. Ostermeier,et al.  Diagnosing male factor infertility using microarrays. , 2003, Journal of andrology.

[14]  P. Khatri,et al.  Spermatozoal RNA profiles of normal fertile men , 2002, The Lancet.

[15]  David A Hildeman,et al.  Determining the Potentiative State of a Chromatin Domain Precipitation Dna Yield a Absorbance B Table 1. Dna Yield and Purity from First and Second Ethanol Precipitations , 1999 .

[16]  S. Krawetz,et al.  Determining the potentiative state of a chromatin domain. , 1997, BioTechniques.

[17]  R. Lilford,et al.  Differential RNA fingerprinting as a tool in the analysis of spermatozoal gene expression. , 1994, Human reproduction.