Abstract 1501: Optimizing small RNAseq of archived serum samples from the Janus Serum Bank - a search for cancer biomarkers

Abstract

Abstract Introduction miRNAs are a distinct class of small non-coding RNA molecules that regulate the expression of protein coding genes. They are present and stable in the cell-free body fluids such as plasma, serum and urine. The Janus Serum Bank contains 317 000 serum samples and more than 61 000 incident cancer cases diagnosed during follow-up. It is well-suited for studies of miRNAs as early cancer biomarkers since samples are collected pre-diagnostically. Aim To establish a high-throughput sequencing approach for obtaining high quality miRNA expression profiles from archived Janus serum samples stored for up to 41 years. Further aims were to assess the quantity and quality of small RNAs in the samples, and establish guidelines for the optimal sequencing depth. Methods Total RNA was extracted from eight pilot samples using Trizol and miRNeasy (Qiagen) columns. Sequencing libraries were made using ScriptMiner (EpiCentre) and the samples were sequenced on one HiSeq (Illumina) lane. After adaptor removal the sequences were aligned unguided to the genome. Reads mapping to mirBASE and Ensembl smallRNA features were counted and sequencing saturation curves were calculated. DESeq was used for differential expression analyses. Results High-throughput sequencing results from the Janus samples, with storage times ranging from 13 to 39 years, show that miRNAs are preserved in the samples. High quality miRNA expression profiles, including on average 641 miRNAs per sample, were obtained from our pilot samples. Most of the uniquely mapped reads, on average 84%, are miRNA. We can also identify protein coding RNAs and other groups of small RNA, on average respectively 9% and 6% of the uniquely mapped reads. The preliminary results from sequencing saturation curves, tracking miRNA discovery in each sample as a measure of input reads, have identified a guideline for optimal sequencing depth. Conclusion and future perspectives Here we have shown that the stability, quantity and quantity of long-term stored sera from the Janus Serum Bank are sufficient for obtaining high-quality miRNA expression profiles. High-throughput sequencing approach will be used in the search for miRNA expression profiles as early cancer biomarkers for early diagnosis, improved therapy and surveillance of cancer. Citation Format: Trine B. Rounge, Robert Lyle, Marianne Lauritzen, Kristina Kjaerheim, Tom Grotmol, Tom K. Grimsrud, Randi E. Gislefoss, Steinar Tretli, Giske Ursin, Hilde Langseth. Optimizing small RNAseq of archived serum samples from the Janus Serum Bank - a search for cancer biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1501. doi:10.1158/1538-7445.AM2014-1501