Microarray analysis of human spermatogenic dysfunction

We investigated testicular biopsies from 28 men which presented with full spermatogenesis (Johnsen Score 10, 12 samples), arrest at the spermatid stage (Johnsen Score 8, 6 samples), arrest at spermatocyte stage (Johnsen Score 5, 5 samples) and Sertoli-cell-only syndrom (Johnsen Score 2, 5 samples). Testicular biopsies were obtained by the "sandwich" method and conserved in RNALater (Ambion) during surgery. Keywords: spermatogenesis germ cell differentiation Keywords: disease state analysis Total RNA was extracted from the biopsies by desintegration in RNAPure (Biozym) and post-cleanup with RNEasy columns (Qiagen). 2 ug of total RNA were labeled with the Amersham Codelink Expression Kit according to the manufacturers protocol, obtaining 40-80 ug of Biotin-labeled cRNA. 10 ug of fragmented cRNA were hybridized for 16h to Human 20K Codelink microarrays (Amersham), washed and stained with Cy5-Streptavidin according to manufacturers protocols and scanned with an Affymetrix 428 Array Scanner at gain 70. Spots were quantified by Codelink Expression software v4.0.