[One-step real-time fluorescence quantitative PCR for detecting WT1 mRNA expression in leukemia].
OBJECTIVE
To establish a one-step real-time quantitative RT-PCR assay for detecting the expression of WT1 mRNA, which allows detection of the minimal residue disease and prognostic prediction in leukemic patients.


METHODS
WT1 gene fragment was amplified from the RNAs extracted from K562 cells using one-step RT-PCR. The quantitative standard were constructed by pMD 18-T vector cloning, and a Taqman-MGB fluorescent probe and a pair of primers were used to establish the one-step real-time fluorescence quantitative RT-PCR assay for WT1 gene detection. The sensitivity, repeatability and stability of this assay were evaluated and verified.


RESULTS
The sensitivity of this assay reached the 10(-4) level. The standard template of 1.0 x 10(6)-1.0 x 10(2) copies/ml were amplified by the one-step real-time fluorescence quantitative RT-PCR assay , and the Ct value was strongly correlated (r=0.998) to the logarithm of the initial template concentration. The repetition Ct value and both the inter-tube and inter-batch coefficients of variation (CV%) were less than 8%.


CONCLUSIONS
The one-step real-time fluorescence quantitative RT-PCR assay has good sensitivity, repeatability and specificity, and the one-step completion of the reverse transcription and PCR processes may reduce the operational complexities and the possibility of contamination.
Xu
Wen-juan
WJ
Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. xuwenjuan5@yahoo.com.cn
Xu
Bing
B
Li
Bing
B
D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D025721:WT1 Proteins / Q000032:analysis* / Q000378:metabolism
D012333:RNA, Messenger; D025721:WT1 Proteins