Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization
Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization
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DOI：
10.1038/labinvest.3700303
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发表时间：
2005-08-01
影响因子：
5
通讯作者：
Jaggi, R
中科院分区：
文献类型：
--
作者：
Antonov, J;Goldstein, DR;Jaggi, R
定量逆转录酶实时PCR（QRT-PCR）是定量RNA丰度的稳健方法。只要初始RNA是完整的，该方法是高度灵敏和可重复的。然而，由于化学或酶促切割导致的RNA断裂可能会减少含有完整扩增子的RNA分子的数量。因此，可用于扩增的分子数量减少。我们确定了RNA片段化和阈值（Ct值）之间的关系，在随后的QRT-PCR中的四个基因的实验模型的完整和部分水解的RNA来自细胞系，我们描述了RNA的完整性，扩增子的大小和Ct值之间的关系，在这个生物均质系统。我们证明，降解相关的位移的Ct值可以通过计算测试基因和几个控制基因的平均值之间的Δ Ct值进行补偿。这些Δ Ct值对RNA的片段化不太敏感，并且不受不同量的输入RNA的影响。通过比较完整和部分降解RNA中较大基因组的Ct值证明了该程序的可行性。我们比较了来自保存完好的肿瘤材料的完整RNA和来自福尔马林固定的石蜡包埋（FFPE）相同肿瘤样品的片段化RNA的Ct值。我们证明，基因表达的相对丰度可以基于FFPE材料，即使样品中RNA的量和片段化的程度未知。
Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.