PMID- 26294305
OWN - NLM
STAT- MEDLINE
DCOM- 20160608
LR  - 20181113
IS  - 1559-0305 (Electronic)
IS  - 1073-6085 (Linking)
VI  - 57
IP  - 10
DP  - 2015 Oct
TI  - Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with 
      Higher Efficiency and Accuracy.
PG  - 939-46
LID - 10.1007/s12033-015-9885-y [doi]
AB  - Understanding of the functional significance of microRNAs (miRNAs) requires 
      efficient and accurate detection method. In this study, we developed an improved 
      miRNAs quantification system based on quantitative real-time polymerase chain 
      reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey 
      the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and 
      mature miRNAs. Instead of relative quantification method, we quantified the 
      pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I 
      fluorescence. This improvement corrected for the inaccuracy caused by the 
      differences in amplicon length and PCR efficiency. We also used SYBR Green method 
      to quantify mature miRNAs based on the stem-loop qRT-PCR method. We extended the 
      pairing part of the stem-loop reverse transcript (RT) primer from 6 to 11 bp, 
      which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The 
      performance of the improved RT primer was tested using synthetic mature miRNAs 
      and tissue RNA samples. Results showed that the improved RT primer demonstrated 
      dynamic range of seven orders of magnitude and sensitivity of detection of 
      hundreds of copies of miRNA molecules.
FAU - Tong, Li
AU  - Tong L
AD  - The College of Environmental Science & Engineering, Donghua University, 2999 
      North Renmin Road, Songjiang, 200237, Shanghai, China.
FAU - Xue, Huihui
AU  - Xue H
FAU - Xiong, Li
AU  - Xiong L
FAU - Xiao, Junhua
AU  - Xiao J
FAU - Zhou, Yuxun
AU  - Zhou Y
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Switzerland
TA  - Mol Biotechnol
JT  - Molecular biotechnology
JID - 9423533
RN  - 0 (MicroRNAs)
RN  - 0 (RNA Precursors)
RN  - 0 (RNA primers)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Animals
MH  - Inverted Repeat Sequences
MH  - Mice
MH  - MicroRNAs/*analysis/chemistry
MH  - RNA/genetics
MH  - RNA Precursors/analysis/chemistry
MH  - Real-Time Polymerase Chain Reaction/*methods
MH  - Reverse Transcriptase Polymerase Chain Reaction/*methods
EDAT- 2015/08/22 06:00
MHDA- 2016/06/09 06:00
CRDT- 2015/08/22 06:00
PHST- 2015/08/22 06:00 [entrez]
PHST- 2015/08/22 06:00 [pubmed]
PHST- 2016/06/09 06:00 [medline]
AID - 10.1007/s12033-015-9885-y [doi]
PST - ppublish
SO  - Mol Biotechnol. 2015 Oct;57(10):939-46. doi: 10.1007/s12033-015-9885-y.