Argyropoulos Dimitris, Psallida Charoula, Spyropoulos Caroline G
Institute of Biotechnology, National Agricultural Research Foundation, Athens, Greece.
FEBS J. 2006 Feb;273(4):770-7. doi: 10.1111/j.1742-4658.2006.05109.x.
A generic sample normalization method applicable in relative comparison of mRNAs quantified with real-time polymerase chain reaction (PCR) is proposed. The method was applied in samples obtained from tomato seeds after osmopriming and aging treatments and from untreated seeds at early imbibition stage, when seeds had not completed germination. Normalization in sample variations was accomplished by detecting synthetic DNA sequences tailing cDNA after second strand reverse transcription synthesis, while the use of the common normalizer GAPDH proved unreliable. Results, obtained from the new method and having a standard error less than 10%, verified the expression profile of a germination-specific mannanase gene that was closely recorded at different time intervals in relation to seed germination.
提出了一种适用于实时聚合酶链反应（PCR）定量的mRNA相对比较的通用样本归一化方法。该方法应用于经渗透引发和老化处理后的番茄种子以及早期吸水阶段未处理种子（此时种子尚未完成萌发）所获得的样本。通过检测第二链逆转录合成后尾随cDNA的合成DNA序列来实现样本变异的归一化，而使用常见的标准化基因甘油醛-3-磷酸脱氢酶（GAPDH）被证明是不可靠的。从新方法获得的结果，其标准误差小于10%，验证了一个与种子萌发密切相关的、在不同时间间隔被密切记录的萌发特异性甘露聚糖酶基因的表达谱。
