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.
authors: Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors: Daniel H
authors: DH
authors: Buitrago
authors: Department of Thoracic Surgery, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
authors: Santosh K
authors: SK
authors: Patnaik
authors: Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America; Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors: Kyuichi
authors: K
authors: Kadota
authors: Department of Thoracic Surgery, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
authors: Eric
authors: E
authors: Kannisto
authors: Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors: David R
authors: DR
authors: Jones
authors: Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America; Center of Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors: Prasad S
authors: PS
authors: Adusumilli
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics*; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA
chemical_list: D035683:MicroRNAs