Exogenous spike-in mouse RNAs for accurate differential gene expression analysis in barley using RT-qPCR.

IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Biology Methods and Protocols Pub Date : 2023-11-28 eCollection Date: 2023-01-01 DOI:10.1093/biomethods/bpad034
Marcus A Vinje, David A Friedman
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Abstract

Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) followed by the 2-ΔΔCt method is the most common way to measure transcript levels for relative gene expression assays. The quality of an RT-qPCR assay is dependent upon the identification and validation of reference genes to normalize gene expression data. The so-called housekeeping genes are commonly used as internal reference genes because they are assumed to be ubiquitously expressed at stable levels. Commonly, researchers do not validate their reference genes but rely on historical reference genes or previously validated genes from an unrelated experiment. Using previously validated reference genes to assess gene expression changes occurring during malting resulted in extensive variability. Therefore, a new method was tested and validated to circumvent the use of internal reference genes. Total mouse RNA was chosen as the external reference RNA and a suite of primer sets to putatively stable mouse genes was created to identify stably expressed genes for use as an external reference gene. cDNA was created by co-amplifying total mouse RNA, as an RNA spike-in, and barley RNA. When using the external reference genes to normalize malting gene expression data, standard deviations were significantly reduced and significant differences in transcript abundance were observed, whereas when using the internal reference genes, standard deviations were larger with no significant differences seen. Furthermore, external reference genes were more accurate at assessing expression levels in malting and developing grains, whereas the internal reference genes overestimated abundance in developing grains and underestimated abundance in malting grains.

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利用 RT-qPCR 对大麦中的外源性尖峰小鼠 RNA 进行精确的差异基因表达分析。
反转录酶定量聚合酶链反应(RT-qPCR)后的 2-ΔΔCt 法是相对基因表达检测中测量转录本水平的最常用方法。RT-qPCR 检测的质量取决于参考基因的鉴定和验证,以便对基因表达数据进行归一化。所谓的看家基因通常被用作内部参考基因,因为它们被认为以稳定的水平普遍表达。通常情况下,研究人员并不验证他们的参考基因,而是依赖于历史参考基因或先前在无关实验中验证过的基因。使用以前验证过的参考基因来评估发芽过程中发生的基因表达变化会产生很大的变异。因此,我们测试并验证了一种新方法,以避免使用内部参考基因。我们选择小鼠总 RNA 作为外部参考 RNA,并创建了一套引物组,用于鉴定小鼠基因是否稳定表达,以用作外部参考基因。使用外部参考基因对麦芽基因表达数据进行归一化处理时,标准偏差显著降低,转录本丰度也出现了显著差异;而使用内部参考基因时,标准偏差较大,且未出现显著差异。此外,外部参考基因在评估发芽谷物和发育中谷物的表达水平方面更为准确,而内部参考基因则高估了发育中谷物的丰度,低估了发芽谷物的丰度。
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来源期刊
Biology Methods and Protocols
Biology Methods and Protocols Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
3.80
自引率
2.80%
发文量
28
审稿时长
19 weeks
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