A novel method to monitor the expression of microRNAs.
The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with important roles in the regulation of gene expression. Although a large number of miRNAs have been identified in a variety of eukaryotic systems, the function of the vast majority of these molecules remains unknown. To study the functions of miRNAs, it is crucial to determine their spatial and temporal expression patterns. Although there are some existing methods that can analyze the expression of miRNAs, it is not an easy task for routine gene-expression studies. In this study, we have established a simple method to detect the expression of mature miRNAs. Total RNA was polyadenylated by poly(A) polymerase, and then cDNA was synthesized by a specific reverse transcriptase (RT) primer and reverse transcriptase using the poly(A)-tailed total RNA as templates. The expression of several mature miRNAs was assayed by this method. The expression profile of two miRNAs, determined by the polymerase chain reaction (PCR) assay, was identical to that determined by Northern blotting. All these data show that the poly(A)-tailed RT-PCR is a convenient method to detect the expression of miRNAs.
Fu
Han-Jiang
HJ
Beijing Institute of Radiation Medicine, Beijing 100850, People's Republic of China.
Zhu
Jie
J
Yang
Ming
M
Zhang
Zhuo-Yuan
ZY
Tie
Yi
Y
Jiang
Hong
H
Sun
Zhi-Xian
ZX
Zheng
Xiao-Fei
XF
D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D011061:Poly A / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D035683:MicroRNAs; D011061:Poly A