title: Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens.
abstract: OBJECTIVE
  Adenocarcinoma in situ (AIS) is an intermediate step in the progression of normal lung tissue to invasive adenocarcinoma. However, molecular mechanisms underlying this progression remain to be fully elucidated due to challenges in obtaining fresh clinical samples for downstream analyses. Formalin fixation and paraffin embedding (FFPE) is a tissue preservation system widely used for long-term storage. Until recently, challenges in working with FFPE precluded using new RNA sequencing technologies (RNA-seq), which would help clarify key pathways in cancer progression. Also, isolation techniques including laser-capture micro-dissection provide the ability to select histopathologically distinct tissues, allowing researchers to study transcriptional variations between tightly juxtaposed cell and tissue types.
  
  
  METHODS
  Utilizing these technologies and new alignment tools we examined differential expression of long intergenic non-coding RNAs (lincRNAs) and mRNAs across normal, AIS and invasive adenocarcinoma samples from six patients to identify possible markers of lung cancer progression.
  
  
  RESULTS
  RNA extracted and sequenced from these 18 samples generated an average of 198 million reads per sample. After alignment and filtering, uniquely aligned reads represented an average 35% of the total reads. We detected differential expression of a number of lincRNAs and mRNAs when comparing normal to AIS, or AIS to invasive adenocarcinoma. Of these, 5 lincRNAs and 31 mRNAs were consistently up- or down-regulated from normal to AIS and more so to invasive carcinoma. We validated the up-regulation of two mRNAs and one lincRNA by RT-qPCR as proof of principle.
  
  
  CONCLUSIONS
  Our findings indicate a potential role of not only mRNAs, but also lincRNAs in the progression to invasive adenocarcinoma. We anticipate that these findings will lay the groundwork for future experimental studies of candidate RNAs from FFPE to identify their functional roles in lung cancer.
authors.affiliation: Department of Family Medicine and Community Health, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Morton
authors.affiliation: Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Bai
authors.affiliation: Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA.|Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Callie R
authors.initials: CR
authors.lastname: Merry
authors.affiliation: Department of Surgery, University Hospitals Case Medical Center, Cleveland, OH, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Linden
authors.affiliation: Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA.|Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Ahmad M
authors.initials: AM
authors.lastname: Khalil
authors.affiliation: Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.|Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Rom S
authors.initials: RS
authors.lastname: Leidner
authors.affiliation: Department of Family Medicine and Community Health, Case Western Reserve University, Cleveland, OH, USA.|Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Thompson
mesh_terms: D065311:Adenocarcinoma in Situ / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D018450:Disease Progression; D005557:Formaldehyde / Q000737:chemistry; D020869:Gene Expression Profiling; D063990:Gene Ontology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D061346:Laser Capture Microdissection; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D016612:Paraffin Embedding; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017423:Sequence Analysis, RNA; D016707:Tissue Fixation; D059467:Transcriptome
chemical_list: D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D005557:Formaldehyde
keywords: Adenocarcinoma in situ of the lung; Formalin-fixed paraffin embedded (FFPE); Gene expression; Invasive lung adenocarcinoma; Laser capture microdissection (LCM); RNA-Seq; lincRNA
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