[Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction].
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.
Gong
Bang-dong
BD
Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University, Shanghai 200025, China.
Xie
Qing
Q
Wang
Lin
L
Xiang
Xiao-gang
XG
Lin
Lan-yi
LY
Zhao
Gang-de
GD
Wang
Hui
H
Yu
Hong
H
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
D017931:DNA Primers; D035683:MicroRNAs