title: Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer.
abstract: OBJECTIVE
  MicroRNAs (miRNAs) play important roles in the initiation and progression of lung cancer. Measuring miRNA expression levels in sputum could provide a potential approach for the diagnosis of lung cancer. The emerging digital PCR is a straightforward technique for precise, direct, and absolute quantification of nucleic acids. The objective of the study was to investigate whether digital PCR could be used to quantify miRNAs in sputum for lung cancer diagnosis.
  
  
  METHODS
  We first determined and compared dynamic ranges of digital PCR and conventional quantitative reverse transcriptase PCR (qRT-PCR) for miRNA quantification using RNA isolated from sputum of five healthy individuals. We then used digital PCR to quantify copy number of two lung cancer-associated miRNAs (miR-31 and miR-210) in 35 lung cancer patients and 40 cancer-free controls.
  
  
  RESULTS
  Copy number of the miRNAs measured by digital PCR displayed a linear response to input cDNA amount in a twofold dilution series over seven orders of magnitude. miRNA quantification determined by digital PCR assay was in good agreement with that obtained from qRT-PCR analysis in sputum. Furthermore, combined quantification of miR-31 and miR-210 copy number by using digital PCR in sputum of the cases and controls provided 65.71 % sensitivity and 85.00 % specificity for lung cancer diagnosis.
  
  
  CONCLUSIONS
  As digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number in sputum for lung cancer diagnosis.
authors.affiliation: Department of Pathology, The University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, 10 South Pine Street, MSTF 7th Floor, Baltimore, MD 21201-1192, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Pathology, The University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, 10 South Pine Street, MSTF 7th Floor, Baltimore, MD 21201-1192, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Pathology, The University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, 10 South Pine Street, MSTF 7th Floor, Baltimore, MD 21201-1192, USA.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Guarnera
authors.affiliation: Division of Biostatistics, The University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: HongBin
authors.initials: H
authors.lastname: Fang
authors.affiliation: Division of Biostatistics, The University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Cai
authors.affiliation: Department of Pathology, The University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, 10 South Pine Street, MSTF 7th Floor, Baltimore, MD 21201-1192, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Jiang
mesh_terms: D000368:Aged; D016022:Case-Control Studies; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013183:Sputum / Q000737:chemistry*
chemical_list: D035683:MicroRNAs