title: [Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction].
abstract: OBJECTIVE
  To establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells.
  
  
  METHODS
  Total RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software.
  
  
  RESULTS
  Microarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot.
  
  
  CONCLUSIONS
  Stem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.
authors.affiliation: Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University, Shanghai 200025, China.
authors.forename: Bang-dong
authors.initials: BD
authors.lastname: Gong
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Xiao-gang
authors.initials: XG
authors.lastname: Xiang
authors.forename: Lan-yi
authors.initials: LY
authors.lastname: Lin
authors.forename: Gang-de
authors.initials: GD
authors.lastname: Zhao
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D035683:MicroRNAs