Small RNA sequencing for profiling MicroRNAs in long-term preserved formalin-fixed and paraffin-embedded non-small cell lung cancer tumor specimens Journal Article

   

Authors:	Buitrago, D. H.; Patnaik, S. K.; Kadota, K.; Kannisto, E.; Jones, D. R.; Adusumilli, P. S.
Article Title:	Small RNA sequencing for profiling MicroRNAs in long-term preserved formalin-fixed and paraffin-embedded non-small cell lung cancer tumor specimens
Abstract:	Background: The preservation of microRNAs in formalin-fixed and paraffin-embedded (FFPE) tissue makes them particularly useful for biomarker studies. The utility of small RNA sequencing for microRNA expression profiling of FFPE samples has yet to be determined. Methods: Total RNA was extracted from de-paraffinized and proteinase K-treated FFPE specimens (15-20 years old) of 8 human lung adenocarcinoma tumors by affinity chromatography on silica columns. MicroRNAs in the RNA preparations were quantified by the Illumina HiSeq 2000 sequencing platform with sequencing libraries prepared with the TruSeq Small RNA Sample Preparation Kit (version 2.0) to obtain unpaired reads of 50 b for small RNA fragments. MicroRNAs were also quantified using Agilent Human miRNA (release 16.0) microarrays that can detect 1,205 mature microRNAs and by quantitative reverse transcription (RT)-PCR assays. Results: Between 9.1-16.9 million reads were obtained by small RNA sequencing of extracted RNA samples. Of these, only 0.6-2.3% (mean = 1.5%) represented microRNAs. The sequencing method detected 454-625 microRNAs/sample (mean = 550) compared with 200-349 (mean = 286) microRNAs detected by microarray. In Spearman correlation analyses, the average correlation coefficient for the 126 microRNAs detected in all samples by both methods was 0.37, and >0.5 for 63 microRNAs. In correlation analyses of the sequencing- and RT-PCR-based measurements, the coefficients were 0.19-0.95 (mean = 0.73) and >0.7, respectively, for 7 of 9 examined microRNAs. The average inter-replicate Spearman correlation coefficient for the sequencing method was 0.81. Conclusions: Small RNA sequencing can be used to obtain microRNA profiles of FFPE tissue specimens with performance characteristics similar to those of microarrays, in spite of the fragmentation of ribosomal and messenger RNAs that reduces the method's informative capacity. The accuracy of the method can conceivably be improved by increasing sequencing depth and/or depleting FFPE tissue RNAs of ribosomal RNA fragments. © 2015 Buitrago et al.
Keywords:	adolescent; adult; clinical article; controlled study; human tissue; protein expression; unclassified drug; reverse transcription polymerase chain reaction; microrna; microarray analysis; formaldehyde; correlational study; tissue preservation; paraffin; rna sequence; non small cell lung cancer; rna analysis; microrna 223; microrna 21; microrna 16; microrna 205; microrna 22; human; article; proteinase k; microrna 146b; microrna 210; microrna 423; microrna 486
Journal Title:	PLoS ONE
Volume:	10
Issue:	3
ISSN:	1932-6203
Publisher:	Public Library of Science
Date Published:	2015-03-26
Start Page:	e0121521
Language:	English
DOI:	10.1371/journal.pone.0121521
PROVIDER:	scopus
PMCID:	PMC4374839
PUBMED:	25812157
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-84926291228&partnerID=40&md5=33bf79516a0ef027a7c859c8bac0994b
Notes:	Export Date: 4 May 2015 -- Source: Scopus

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