Effective Alu Repeat Based RT-Qpcr Normalization in Cancer Cell Perturbation Experiments

Background
Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.
Results
The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5).
Conclusions
We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.

背景
逆转录定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction，RT-qPCR）检测信使RNA（messenger RNA，mRNA）水平是众多实验室的常规实验方法。选取特定的mRNA组合作为内参参考基因，被认为是标准化RT-qPCR数据的最优策略。参考基因的合理筛选是一项关键问题，尤其对于经历多种体外操作的癌细胞而言，此类操作可能引发基因表达水平的剧烈改变，即便此前被认为是稳定的参考基因也难以幸免。本研究针对19项实验，评估了11种常用参考基因作为内参的表达水平，这些实验涉及的细胞系包括神经母细胞瘤、T细胞急性淋巴细胞白血病（T-ALL）、黑色素瘤、乳腺癌、非小细胞肺癌（NSCL）、急性髓系白血病（AML）、前列腺癌、结直肠癌及宫颈癌的细胞系，所有细胞系均接受了各类扰动处理。
结果
本研究借助qbase+软件包中的geNorm算法，依据表达稳定性对候选参考基因进行排序。研究发现，多数候选参考基因的稳定性在扰动实验中差异显著。表达的Alu重复序列的表达水平相对稳定，不受实验条件影响。此类Alu重复序列在所有扰动实验中均位列最优参考检测方法之列，且平均表达稳定性值处于可接受范围（M<0.5）。
结论
本研究建议，在开展癌细胞扰动实验时，可采用Alu重复序列作为参考检测方法。