Real-time polymerase chain reaction microRNA detection based on enzymatic stem-loop probes ligation.
MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biological processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addition, a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examined as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary laboratory studies and clinical diagnoses.
Li
Juan
J
School of Electronic and Information Engineering, Beihang University, Beijing, China, 100191.
Yao
Bo
B
Huang
Huang
H
Wang
Zhao
Z
Sun
Changhong
C
Fan
Yu
Y
Chang
Qing
Q
Li
Shaolu
S
Wang
Xiang
X
Xi
Jianzhong
J
D000818:Animals; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D005784:Gene Amplification; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*
D015342:DNA Probes; D035683:MicroRNAs; C517731:Mirn1 microRNA, mouse; D011088:DNA Ligases