Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis.
Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis.
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在COVID-19和粘膜细胞增多中选择理想的参考基因进行基因表达分析。
DOI：
10.1128/spectrum.01656-22
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发表时间：
2022-12-21
影响因子：
3.7
通讯作者：
Saini, Vikram
中科院分区：
文献类型：
--
作者：
Kumar, Sunil;Ahmad, Ayaan;Kushwaha, Namrata;Shokeen, Niti;Negi, Sheetal;Gautam, Kamini;Singh, Anup;Tiwari, Pavan;Garg, Rakesh;Agarwal, Richa;Mohan, Anant;Trikha, Anjan;Thakar, Alok;Saini, Vikram
在实时定量PCR（qRT-PCR）过程中参考基因的选择对于确定准确和可靠的mRNA表达至关重要。尽管如此，没有一项研究调查了候选参考基因的表达稳定性，以确定其作为SARS-CoV-2感染或COVID-19相关毛霉菌病（CAM）内部对照的适用性。使用qRT-PCR，我们确定了9种最常用的管家基因的表达稳定性，即TATA盒结合蛋白（TBP）、亲环蛋白（CypA）、β-2-微球蛋白（B2M）、18S rRNA（18S）、过氧化物酶体增殖物激活受体γ（PPARG）共激活因子1 α（PGC-1 α）、葡萄糖醛酸酶β（GUSB）、次黄嘌呤磷酸核糖基转移酶1（HPRT-1）、β-肌动蛋白、和甘油醛-3-磷酸脱氢酶（GAPDH）在各种严重程度（无症状、轻度、中度和重度）的COVID-19患者和CAM患者中的作用。我们使用统计算法（delta-CT [阈值循环]、Normalization、BestKeeper、GeNorm和Referral）选择最合适的参考基因，并观察到临床严重程度深刻影响参考基因的表达稳定性。CypA表现出与疾病严重程度无关的最一致的表达，并成为COVID-19和CAM中最合适的参考基因。顺便说一句，GAPDH，最常用的参考基因，表现出最大的表达变化，并出现作为最不合适的。接下来，我们使用CypA和GAPDH作为内部对照测定了核因子红细胞2相关因子2（NRF2）、白细胞介素-6（IL-6）和IL-15的表达，并显示CypA标准化表达与这些基因基于RNA测序的表达匹配良好。此外，IL-6表达与IL-6和C-反应蛋白（炎症标志物）的血浆水平相关性良好。总之，GAPDH是COVID-19和CAM中最不合适的参考基因，CypA是最合适的参考基因。这些结果突出了由于疾病严重程度导致的管家基因的表达变异性，并为在其他慢性疾病中鉴定适当的参考基因提供了强有力的理论基础。基因表达研究对于开发新的诊断、治疗和预后模式至关重要。然而，表达的准确测定需要用参考基因进行数据归一化，其表达在不同疾病阶段之间不变化。参考基因的错误鉴定会产生不准确的结果。不幸的是，尽管COVID-19的全球影响以及对更好治疗的迫切需求未得到满足，但没有一项研究调查了整个疾病谱中管家基因的表达稳定性，以确定其作为内部对照的适用性。我们的研究确定CypA和TBP是COVID-19和CAM的两个最合适的参考基因。此外，在COVID-19研究中最常用的参考基因GAPDH被证明是最不合适的。这项工作填补了该领域的一个重要空白，并有望促进确定基因的准确表达，以催化开发新的分子诊断和治疗方法，改善患者护理。
Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), β-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), β-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.
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影响因子：
4.8
作者：
Geyer, Henriette;Bauer, Michael;Mankertz, Annette
通讯作者：
Mankertz, Annette
DOI：
10.1016/s1473-3099(20)30486-2
发表时间：
2021-03
期刊：
The Lancet. Infectious diseases
影响因子：
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作者：
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通讯作者：
Woods CW
影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
DOI：
10.1073/pnas.1121249109
发表时间：
2012-10-16
影响因子：
11.1
作者：
Lam, Lucia L.;Emberly, Eldon;Kobor, Michael S.
通讯作者：
Kobor, Michael S.
影响因子：
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作者：
Ahn K;Huh JW;Park SJ;Kim DS;Ha HS;Kim YJ;Lee JR;Chang KT;Kim HS
通讯作者：
Kim HS