Identification of best housekeeping genes for the normalization of RT-qPCR in human cell lines

Braga, Letícia da Conceição; Gonçalves, Bryan Ôrtero Perez; Coelho, Pollyanne Lacerda; Filho, Agnaldo Lopes da Silva; Silva, Luciana Maria

doi:10.1016/j.acthis.2021.151821

Cited by 10 publications

(7 citation statements)

References 31 publications

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“…It is worth noting that although many studies have reported the use of only one HKG, a normalization strategy based on a single reference gene can lead to erroneous results. Therefore, some suggest using at least two reference genes to avoid significant errors [9].…”

Section: Discussionmentioning

confidence: 99%

“…ACTB and GAPDH were selected for this project. The choice was made due to the fact that these genes belong to the classic genes of basic metabolism, historically considered to be expressed stably in human cells [9], and are already used as reference genes in molecular testing of nasopharyngeal and oropharyngeal swabs by some scientists [10].…”

Section: Introductionmentioning

confidence: 99%

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What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

et al. 2023

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Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955–0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

et al. 2023

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“…In general, constitutively expressed housekeeping genes are used as reference genes, such as actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA, translation initiation factor, tubulin and ubiquitin [7][8][9]. However, many studies have shown that some of these genes are not always stable and their expression levels vary greatly under speci c experimental conditions [6,10,11].…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, normalization is necessary to correct for variations in template quantity. Reference genes are used for the normalization of gene expression because of the stability of expression levels among different tissues, different developmental stages, or under various treatments [5].In general, constitutively expressed housekeeping genes are used as reference genes, such as actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA, translation initiation factor, tubulin and ubiquitin [7][8][9]. However, many studies have shown that some of these genes are not always stable and their expression levels vary greatly under speci c experimental conditions [6,10,11].…”

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confidence: 99%

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Chen

Wang

Yang

et al. 2023

Preprint

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Background Real-time quantitative PCR(RT-qPCR) is a crucial and widely used method for gene expression analysis. Selecting suitable reference genes is extremely important for the accuracy of RT-qPCRresults. Commonly used reference genes are not always stable in various organisms or under different environmental conditions. With the increasing application of high-throughput sequencing, transcriptome analysis has become an effective method for identifying novel stable reference genes. Results In this study, we identified candidate reference genes based on transcriptome data covering embryos and larvae of early development, normal adult tissues, and the hindgut under sulfide stress using the CV method in the echiuran Urechis unicinctus, resulting in 2093 (4.84%), 2534 (6%), and 9648 (24.94%) candidate reference genes, respectively. GO and KEGG enrichment analyses revealed that the candidate reference genes were significantly enriched in cellular metabolic process, protein metabolic process and ribosome in early development and normal adult tissues as well as in cellular localization and endocytosis in the hindgut undersulfide stress. Subsequently, ten genes including five new candidate reference genes and five commonly used reference genes, were validated by RT-qPCR. The expression stability of the ten genes was analyzed using four methods (geNorm, NormFinder, BestKeeper, and ∆Ct). The comprehensive results indicated that the new candidate reference genes were more stable than most commonly used reference genes. The commonly used ACTB was the most unstable gene. The candidate reference genes STX12, EHMT1, and LYAG were the most stable genes in early development, normal adult tissues, and hindgut undersulfide stress, respectively. The FPKM of the transcriptome data was significantly negatively correlated with the Ct values of RT-qPCR (Ct = − 0.002518 FPKM + 26.63), which made it possible to estimate the Ct value before RT-qPCR using transcriptome data. Conclusion Our study is the first to select reference genes for RT-qPCR from transcriptome data in Echiura and provides important information for future gene expression studies in U. unicinctus.

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“…In general, constitutively expressed housekeeping genes are used as reference genes, such as actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase, ribosomal RNA, translation initiation factor, tubulin, and ubiquitin [ 7 – 9 ]. However, many studies have shown that some of these genes are not always stable and their expression levels vary greatly under specific experimental conditions [ 6 , 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Chen,

Wang,

Yang

et al. 2023

BMC Genomics

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Background Real-time quantitative PCR (RT-qPCR) is a crucial and widely used method for gene expression analysis. Selecting suitable reference genes is extremely important for the accuracy of RT-qPCR results. Commonly used reference genes are not always stable in various organisms or under different environmental conditions. With the increasing application of high-throughput sequencing, transcriptome analysis has become an effective method for identifying novel stable reference genes. Results In this study, we identified candidate reference genes based on transcriptome data covering embryos and larvae of early development, normal adult tissues, and the hindgut under sulfide stress using the coefficient of variation (CV) method in the echiuran Urechis unicinctus, resulting in 6834 (15.82%), 7110 (16.85%) and 13880 (35.87%) candidate reference genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the candidate reference genes were significantly enriched in cellular metabolic process, protein metabolic process and ribosome in early development and normal adult tissues as well as in cellular localization and endocytosis in the hindgut under sulfide stress. Subsequently, ten genes including five new candidate reference genes and five commonly used reference genes, were validated by RT-qPCR. The expression stability of the ten genes was analyzed using four methods (geNorm, NormFinder, BestKeeper, and ∆Ct). The comprehensive results indicated that the new candidate reference genes were more stable than most commonly used reference genes. The commonly used ACTB was the most unstable gene. The candidate reference genes STX12, EHMT1, and LYAG were the most stable genes in early development, normal adult tissues, and hindgut under sulfide stress, respectively. The log2(TPM) of the transcriptome data was significantly negatively correlated with the Ct values of RT-qPCR (Ct = − 0.5405 log2(TPM) + 34.51), which made it possible to estimate the Ct value before RT-qPCR using transcriptome data. Conclusion Our study is the first to select reference genes for RT-qPCR from transcriptome data in Echiura and provides important information for future gene expression studies in U. unicinctus.

show abstract

Identification of best housekeeping genes for the normalization of RT-qPCR in human cell lines

Cited by 10 publications

References 31 publications

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

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