在 SK-BR-3 乳腺癌細胞系中鑒定用于 qPCR 標(biāo)準(zhǔn)化的新型內(nèi)源性對照

Identification of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line

Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use. Hence, in the presentstudy we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breastcancer cell line. Transcriptomic data of 82 HER2-E breast cancer samples from TCGA database were analyzed to identify twelve novel genes with stable expression. Additionally, thirteen RGs from the literature were analyzed. The expression variations of the candidate genes were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia. Our results indicate that HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimiz。ing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage the use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line.

T75細胞培養(yǎng)瓶

使用內(nèi)部對照或參考基因 (RG) 對基因表達進行標(biāo)準(zhǔn)化已成為標(biāo)準(zhǔn)化逆轉(zhuǎn)錄定量聚合酶鏈反應(yīng) (RT-qPCR) 技術(shù)變化的首選方法。傳統(tǒng)上，ACTB和GAPDH盡管文獻中的證據(jù)不鼓勵使用它們，但已被用作參考基因。因此，在本研究中，我們在富含 HER2 的乳腺癌細胞系 SK-BR-3 中鑒定并研究了新的參考基因。對來自 TCGA 數(shù)據(jù)庫的 82 個 HER2-E 乳腺癌樣本的轉(zhuǎn)錄組數(shù)據(jù)進行了分析，以確定十二個具有穩(wěn)定表達的新基因。此外，還分析了文獻中的 13 個 RG。在兩個平行培養(yǎng)物 S1 和 S2 中以及在急性和慢性缺氧條件下，使用各種算法研究了候選基因的表達變化超過 5 次連續(xù)傳代 (p)。最后，選擇最穩(wěn)定的 RGs 并驗證其在常氧和缺氧條件下三個感興趣基因 (GOI) 的表達正常化。我們的結(jié)果表明HSP90AB1、DAD1、PFN1和PUM1可用于三個（三聯(lián)體）的任意組合，以優(yōu)化 SK-BR-3 細胞系中檢測內(nèi)和檢測間的基因表達差異。此外，我們不鼓勵使用傳統(tǒng)的 RG（ACTB、GAPDH、RPL13A、RNA18S和RNA28S）作為 SK-BR-3 細胞系中 RT-qPCR 的內(nèi)部對照。

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) represents a modified variant of the popular conventional PCR with diverse applications, ranging from functional genomics to molecular medicine, virology, microbiology, and biotechnology. Quantitative PCR-based assays can target both DNA (genome) and RNA (transcriptome), thereby making it an extremely powerful and important technique in molecular diagnostics . While functional genomics deals with understanding the functions and interactions of genes and proteins at a genome-wide level including the role of ligands, receptors, and signaling networks that converge on transcriptional regulation, transcriptomic analysis, deals with ascertaining the functional significance to expression signature changes between tissues, disease states, or treatment . Large-scale analysis of expression patterns is performed by RNA-Seq or high-throughput microarray analysis, however, the findings for individual genes usually are validated by RT-qPCR due to its high sensitivity, specificity, reproducibility, and broad dynamic range.

裝有培養(yǎng)基5層細胞工廠

逆轉(zhuǎn)錄定量聚合酶鏈反應(yīng)(RT-qPCR)代表是流行的傳統(tǒng)PCR的改良變種，應(yīng)用范圍廣泛，ing從功能基因組學(xué)到分子醫(yī)學(xué)、病毒學(xué)、微生物學(xué)、生物技術(shù)nology?；诙?/span>pcr的檢測可以針對DNA(基因組)和RNA (transcriptome)，因此使其成為分子ular診斷中一個非常強大和重要的技術(shù)。而功能基因組學(xué)研究的是功能以及基因和蛋白質(zhì)在全基因組水平上的相互作用，包括lig And、受體和匯聚在轉(zhuǎn)錄調(diào)控上的信號網(wǎng)絡(luò)的作用，轉(zhuǎn)錄組學(xué)分析，確定表達的功能意義組織、疾病狀態(tài)或治療之間的特征變化。大規(guī)模的分析表達模式是通過RNA-Seq或高通量微陣列分析進行的，然而，由于單個基因的發(fā)現(xiàn)通常是通過RT-qPCR來驗證的靈敏度、特異性、重現(xiàn)性、動態(tài)范圍廣.

來源：MDPI  https://www.mdpi.com/2073-4425/12/10/1631