Sonntag Kai-C, Tejada George, Subburaju Sivan, Berretta Sabina, Benes Francine M, Woo Tsung-Ung W
Division of Basic Neuroscience, Department of Psychiatry, Harvard Brain Tissue Resource Center, McLean Hospital/Harvard Medical School, Belmont, Massachusetts, USA.
J Neurochem. 2016 Jul;138(1):53-9. doi: 10.1111/jnc.13637. Epub 2016 May 12.
The RNA integrity number (RIN) is often considered to be a critical measure of the quality of postmortem human brains. However, it has been suggested that RINs do not necessarily reflect the availability of intact mRNA. Using the Agilent bioanalyzer and qRT-PCR, we explored whether RINs provide a meaningful way of assessing mRNA degradation and integrity in human brain samples by evaluating the expression of 3'-5' mRNA sequences of the cytochrome C-1 (CYC1) gene. Analysis of electropherograms showed that RINs were not consistently correlated with RNA or cDNA profiles and appeared to be poor predictors of overall cDNA quality. Cycle thresholds from qRT-PCR analysis to quantify the amount of CYC1 mRNA revealed positive correlations of RINs with amplification of full-length transcripts, despite the variable degree of linear degradation along the 3'-5' sequence. These data demonstrate that in postmortem human brain tissue the RIN is an indicator of mRNA quantity independent of degradation, but does not predict mRNA integrity, suggesting that RINs provide an incomplete measure of brain tissue quality. Quality assessment of postmortem human brains by RNA integrity numbers (RINs) may be misleading, as they do not measure intact mRNAs. We show that the RIN is an indicator of mRNA quantity independent of degradation, but does not predict mRNA integrity, suggesting that RINs provide an incomplete measure of brain tissue quality. Our results resolve controversial assumption on interpreting quality assessments of human postmortem brains by RINs.
RNA完整性数值(RIN)通常被认为是衡量死后人类大脑质量的关键指标。然而,有人提出RIN并不一定反映完整mRNA的可用性。我们使用安捷伦生物分析仪和定量逆转录聚合酶链反应(qRT-PCR),通过评估细胞色素C-1(CYC1)基因3'-5'mRNA序列的表达,探讨RIN是否能为评估人类脑样本中的mRNA降解和完整性提供有意义的方法。电泳图分析表明,RIN与RNA或cDNA图谱并不始终相关,似乎不能很好地预测总体cDNA质量。qRT-PCR分析用于定量CYC1 mRNA量的循环阈值显示,尽管沿着3'-5'序列的线性降解程度不同,但RIN与全长转录本的扩增呈正相关。这些数据表明,在死后人类脑组织中,RIN是独立于降解的mRNA数量指标,但不能预测mRNA完整性,这表明RIN对脑组织质量的衡量是不完整的。通过RNA完整性数值(RIN)对死后人类大脑进行质量评估可能会产生误导,因为它们无法衡量完整的mRNA。我们表明,RIN是独立于降解的mRNA数量指标,但不能预测mRNA完整性,这表明RIN对脑组织质量的衡量是不完整的。我们的结果解决了关于通过RIN解释人类死后大脑质量评估的有争议的假设。