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Sources of Funding: P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation_x000D_ _x000D_ This work was supported in part by the Urology Care Foundation Research Scholar Award Program and AUA New York Section Research Scholar Fund

Introduction

Non-coding RNAs (ncRNAs) are emerging as important but poorly understood regulators of mRNA transcription and translation. However, common library preparation techniques for RNA sequencing selects for coding mRNAs by the presence of a poly-A tail; thus, by excluding non-PolyA ncRNAs, many biologically significant transcripts may be overlooked by using this method. The objective of this study was to evaluate differences in testicular RNA identification using 2 different methods of library preparation: polyA and ribosomal RNA depletion (RibZero) using Illumina kits.

Methods

Total RNA was extracted from 3 human testis samples and processed using two different methods of library preparations: one based on polyA tags, and the other on depletion of ribosomal RNAs. The cDNA libraries were then sequenced at the same depth and annotated to known published databases. Identified transcripts were divided into two groups based on presence in one of the library preparation methods but not the other. Clinical and biological significance of identified genes was examined using the DAVID. Failure of detection of RibZero-only genes by RNAseq using polyA preparation was then confirmed by analyzing results of RNAseq in 64 testicular samples from normal patients, men with sertoli cell only (SCO), early and late maturation arrest and hypospermatogenesis.

Results

61 genes were detected only using polyA method with no detection in RibZero group (p=0.05): 17 of them belonged to small nuclear RNAs (snRNAs), 12 were snRNAs hosting genes, 3 humanin like proteins, and 3 were miRNA hosting genes: MIR137HG, MIR17HG, MIRLET7DHG. Deletion of MIR17HG leads to Feingold syndrome in humans and animal models. 74 genes were identified exclusively in RibZero group and not identified in the polyA group. The top 4 genes identified exclusively by RibZero were TAS2R50, MAGI1-AS, HIST1H3I, HIST1H4K. HIST1H4K was then further analyzed and its expression was highly abundant and specific to pachytene spermatocytes. Important components of the miRNA processing complex: AGO1,2, and 3 were expressed at >2x higher level (p=0.03) in ribosomal RNA library depletion preparation then polyA prep.

Conclusions

PolyA tail RNA enrichment method fails to adequately detect at least 7% of important RNAs in the human testis. Including ribosomal depletion RNA library preparation in addition to polyA tags enrichment is an important step to more comprehensively evaluate ncRNAs and testicular function.

Funding

P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation_x000D_ _x000D_ This work was supported in part by the Urology Care Foundation Research Scholar Award Program and AUA New York Section Research Scholar Fund