[Detection of miRNA levels in leukemia patients by real-time quantitative PCR].
This study was purposed to establish a method for detecting miRNA expression by using fluorescent quantitative polymerase chain reaction (RQ-PCR), and to investigate the application value of this method in quantitative detection of miRNA. Total RNA was extracted from the peripheral blood or bone marrow of 82 patients with chronic lymphocytic leukemia (CLL) and 70 patients with acute leukemias (AL). Standard curves of U6 and miRNA were constructed from 10-fold serial dilutions of the cDNA, the quantitative detection was performed by using real-time quantitative PCR with SYBR Green by Roche Light Cycler. U6 RNA was used as the reference, the relative expression levels of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in patients with CLL, while miR-128-1, miR-223, let-7b, miR-155 and miR-181a in patients with AL were analyzed by 2((-DeltaDeltaCT)) method. The relative parameters including specificity, linearity range, sensitivity and repeatability were evaluated. The results showed that the RQ-PCR assay could precisely detect the mature miRNA in as few as 10-50 ng of total RNA with the sensitivity of 0.05 ng RNA. Ct values of miRNA and U6 were all within 15 to 30. A good linear relationship (R(2) > 0.980) was obtained from the standard curves, also the efficiency of amplification was above 90%. Only a specific peak was shown on the melting curve diagram. The coefficients of variation (CV) of interrun and intrarun assay was < 4.8% and 6.3% respectively. It is concluded that the RQ-PCR is a sensitive and fast method for the detection of miRNA with its high-flux and low cost, this method will facilitate the research with detection of miRNA.
Zhu
Dan-Xia
DX
Department of Hematology, Nanjing Medical University First Affiliated Hospital, Jiangsu Province People Hospital, Nanjing 210029, Jiangsu Province, China.
Miao
Kou-Rong
KR
Zhu
Yuan-Dong
YD
Zhu
Hua-Yuan
HY
Fan
Lei
L
Liu
Peng
P
Xu
Wei
W
Li
Jian-Yong
JY
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D005260:Female; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055815:Young Adult
D035683:MicroRNAs