State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nan Xin Cun, Beijing 100093, China.
Plant Methods. 2012 Mar 9;8:9. doi: 10.1186/1746-4811-8-9.
Temporal and tissue-specific patterns of gene expression play important roles in functionality of a biological system. Real-time quantitative polymerase chain reaction (qPCR) technique has been widely applied to single gene expressions, but its potential has not been fully released as most results have been obtained as fold changes relative to control conditions. Absolute quantification of transcripts as an alternative method has yet to gain popularity because of unresolved issues.
We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/μl, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA) qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (Ipomoea purpurea, Nicotiana benthamiana) and tissues (petals and leaves).
By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells.
基因表达的时间和组织特异性模式在生物系统的功能中起着重要作用。实时定量聚合酶链反应（qPCR）技术已广泛应用于单个基因表达，但由于大多数结果都是相对于对照条件的倍数变化获得的，其潜力尚未得到充分释放。作为替代方法的转录物绝对定量尚未普及，因为存在未解决的问题。
我们在这里提出了一种解决方案，一种新的程序，可以准确地对从生物样本中常规制备的总 cDNA 进行定量，分辨率约为 70pg/μl，并可靠地估计 picogram cDNA 中的转录本绝对数量。与相对定量相比，基于 cDNA 的绝对（CBA）qPCR 方法对发育和环境变化等因素引起的基因表达变化更敏感。如果目标转录本拷贝数进一步通过参考转录本进行标准化，那么在发育过程中也可以检测到目标基因表达的细胞水平变化模式，正如在这里观察到的跨物种（Ipomoea purpurea、Nicotiana benthamiana）和组织（花瓣和叶片）的情况。
通过允许在实验之间直接比较结果，新程序为在广泛的生命系统和组织中推断基因表达模式打开了一扇窗户。对于不同细胞中基因表达变化的生物学解释，这种比较是迫切需要的。
