实时荧光定量RT-PCR检测血液肿瘤细胞株PAX5 mRNA表达

Detection of PAX5 mRNA expression in haematological tumor cell lines by real-time reverse transcription polymerase chain reaction

目的 建立实时定量逆转录 聚合酶链反应(RT PCR)检测6个血液肿瘤细胞株中PAX5和CD19的 mRNA相对表达量。方法 一系列稀释的反转录自NAMALWA细胞株总RNA的cDNA被用于构建PAX5、CD19 和GAPDH扩增的标准曲线。实验证实靶基因(PAX5和CD19)与管家基因(GAPDH)的扩增效率一致,因此可 以用比较循环数(Ct)法对PAX5和CD19的表达进行相对定量。结果 在NAMALWA(B细胞系)细胞株中, PAX5和CD19mRNA相对表达量分别为2.35%和2.52%;而在其他T细胞和髓系细胞株中几乎不表达。结论 本研究中所建立的实时定量RT PCR能简单、快速、方便地对PAX5和CD19mRNA表达水平进行定量,且适 用于对大量、不同种血液恶性肿瘤患者进行观察研究。

Objective To develop a real-time reverse transcription polymerase chain reaction(RT-PCR) for the relative quantitation of PAX5 and CD19 mRNA expression in 6 haematological tumor cell lines. Methods A serial dilution cDNA reversely transcriped from total RNA in NAMALWA were used to construct standard curves for the PAX5, CD19 and GAPDH amplifications. The amplification efficiencies were identical for both the target gene(PAX5 and CD19) and house keeping gene(GAPDH). Results PAX5 and CD19 mRNA expression level was 2.35% and 2.52% respectively in NAMALWA(B-cell lines), but almost not detectable in other T-and myeloid cell lines. Conclusions The real-time RT-PCR is simple, rapid and convenient for quantification of PAX5 and CD19 mRNA levels, and is suitable for investigation in larger groups of patients with different haematological malignancies.