Department of Hematology, Post Graduate Institute of Medical Education and Research (PGIMER), Sector 12, Chandigarh, 160012, India.
International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
Mol Biol Rep. 2022 Jun;49(6):4841-4848. doi: 10.1007/s11033-022-07337-w. Epub 2022 Mar 27.
The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene.
The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases.
It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.
管家基因 (HKG) 表达的稳定性是定量实时聚合酶链反应 (RQ-PCR) 中准确归一化靶基因表达的绝对前提。在 RQ-PCR 中，广泛使用的归一化方法涉及将靶基因标准化到最稳定的 HKG 对照基因。根据最近的文献，在不同的实验条件下，HKG 表现出上调或下调，从而影响靶基因的基因表达谱，导致错误的结果。这意味着在定量靶基因之前，选择合适的 HKG 并验证 HKG 的表达稳定性非常重要。
本研究旨在分析 6 种不同的 HKG 在 BCR-ABL1 阴性病例中的表达谱和稳定性，并通过多重逆转录聚合酶链反应 (RT-PCR) 对 BCR-ABL1 阳性病例进行验证。本研究选择了 6 种常用的参考基因 (GAPDH、ABL1、RNA18S、ACTB、GUSB 和 EEF2)。对 24 例 BCR-ABL1 阴性病例进行 RQ-PCR，对 24 例 BCR-ABL1 阳性病例进行验证。RefFinder™，一种基于网络的综合软件，用于通过不同的算法检查 HKG 基因的稳定性，并对 BCR-ABL1 阴性病例中的每个 HKG 基因进行综合排名，最终在 BCR-ABL1 阳性病例中进行验证。
研究发现，与最常用的 HKG（GAPDH、EEF2 和 ACTB）相比，RNA18S、ABL1 和 GUSB 是稳定的 HKG，其基因表达的变异性最小。
