title: [Detection of puma mRNA levels by real-time quantitative RT-PCR in chronic lymphocytic leukemia and its clinical significance].
abstract: This study was aimed to investigate the expression level of puma (p53 up-regulated modulator of apoptosis) mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. The puma mRNA expressions in 100 CLL patients and 11 normal controls were measured by relative quantification RT-PCR with fluorescent dye SYBR Green I, the beta-actin was used as internal reference. The difference of puma expression rate between groups with different prognostic factors was described using the Mann-Whitney U test. The relative quantitative value of puma expression was calculated by means of 2 (-ΔCt). The results indicated that the correlation coefficients of the standard curves in qRT-PCR were ≥ 0.99. The coefficients of variations (CV) within group or between groups were < 5%, and the sensitivity reached 10² copies/microg RNA. The median puma mRNA expression level was 1.038 x 10⁻³ (4.106 x 10⁻⁴ - 2.806 x 10⁻³) in CLL patients, which was 1.220 x 10⁻³ (7.233 x 10⁻⁴ - 1.405 x 10⁻³) in normal controls. There was no difference of puma mRNA expression between CLL patients and normal controls (U = 544.5, p = 0.957). Puma expression was significantly correlated with Binet stages (p < 0.001), expression of CD38 (p = 0.002), ZAP-70 protein (p = 0.012), LDH levels (p = 0.009) and beta₂-MG (p = 0.046). The puma expression level in patients with earlier Binet stage (Binet stage A) was obviously higher than that in patients with later Binet stage (Binet stage B, C). The puma expression levels in patients with positive expression of CD38 and ZAP-70 protein, elevating levels of LDH and beta₂-MG were sharply lower than those in patients without above-mentioned unfavorable factors. The puma expression was also correlated with molecular cytogenetic abnormalities, the puma expression levels in patients with trisomy 12 (p = 0.003) and 14q32 translocation (p = 0.045) detected by FISH were significantly lower than those in patients without above-mentioned molecular cytogenetic abnormalities. It is concluded that the qRT-PCR assay is reliable and sensitive. Puma mRNA expression is significantly correlated with a great deal of prognostic factors, and may be a prognostic marker of CLL.
authors: Department of Hematology, Nanjing Medical University First Hospital, Jiangsu Provincial Hospital, Nanjing 210029, Jiangsu Province, China.
authors: Hai-Jia
authors: HJ
authors: Zhu
authors: Wei
authors: W
authors: Xu
authors: Xin
authors: X
authors: Cao
authors: Cheng
authors: C
authors: Fang
authors: Dan-Xia
authors: DX
authors: Zhu
authors: Hua-Jie
authors: HJ
authors: Dong
authors: Dong-Mei
authors: DM
authors: Wang
authors: Chun
authors: C
authors: Qiao
authors: Kou-Rong
authors: KR
authors: Miao
authors: Peng
authors: P
authors: Liu
authors: Jian-Yong
authors: JY
authors: Li
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism; D000368:Aged; D000369:Aged, 80 and over; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D005260:Female; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D051746:ZAP-70 Protein-Tyrosine Kinase; D051997:ADP-ribosyl Cyclase 1