Validation of Suitable Housekeeping Genes for the Normalization of mRNA Expression for Studying Tumor Acidosis

Lemma, Silvia; Avnet, Sofia; Meade, Michael; Chano, Tokuhiro; Baldini, Nicola

doi:10.3390/ijms19102930

Cited by 21 publications

(14 citation statements)

References 32 publications

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“…We quantitatively checked the presence of several genes, in terms of average expression across the entire cell population (expressed as Log10 average TPM) and in terms of number of cells with at least one read mapped on the gene ( Figure 1 C for BE2C, Figure 1 D for Kelly). We checked the expression of four commonly used housekeeping genes: ACTB, GAPDH, B2M and GUSB [ 44 ]. All these genes are highly expressed and could be detected in almost all cells, with the exception of GUSB, detected in only ~50% of both Kelly and BE2C cells.…”

Section: Resultsmentioning

confidence: 99%

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines

et al. 2021

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Neuroblastoma (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.

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Section: Resultsmentioning

confidence: 99%

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines

et al. 2021

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“…It is important to note that once selected, a reference for a given model will not necessarily be selected once again in an experiment repeated for this model after some time, even if the pool of candidate reference genes used is exactly the same. This is because the gene expression pattern at the time point of sample collection depends on the current rate of cell mass growth and on microenvironmental conditions [20, 21].…”

Section: Discussionmentioning

confidence: 99%

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Hoja-Łukowicz

Maciążek

Janik

2021

Preprint

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Melanoma is characterized by a high mutation rate and the proper reference selection is a serious problem. The algorithms commonly used to select the best stable reference gene or pair of genes in RT-qPCR data analysis have their limitations which affects the interpretation of the results and drawing conclusions. For reliable assessment of the B4GALT genes expression changes in melanoma and comparing them to their expression levels in melanocytes, we implemented an innovative GenExpA software. We proposed selection of the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and the set of daughter models assigned to it. The reliability of references is validated by normalizing of target gene expression level through all models and is described by the coherence score. The software performs statistical analyses and generates results in the form of ready-to-use graphs and tables. GenExpA is a promising tool for basic research; it produces analyses of target gene expression in a manner independent of the experimental model and the normalizer. GenExpA and its manual is freely available at https://drive.google.com/drive/folders/1FcpBtEc_bKPvEnM5GcPZerBb_Tw_fCkt?usp=sharing or https://www.sciencemarket.pl/baza-programow-open-source#oferty. Supplemental materials are deposited under DOI number: 10.5281/zenodo.4544296.

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“…However, many studies have provided convincing evidence that the level of transcription of housekeeping genes differs according to the developmental stage and other experimental conditions [27, 28]. For determination of the best candidate reference gene, the following statistical five algorithms were used: NormFinder, geNorm, BestKeeper, ΔCT, and coefficient of variation [29]. However, normalization results, which cannot be solved by the aforementioned algorithms, vary depending on the housekeeping gene used (e.g., GAPDH, HPRT), the expression of which varies during embryonic stem cell differentiation and according to the stage of embryogenesis in specific tissues [14, 16, 30].…”

Section: Discussionmentioning

confidence: 99%

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method

et al. 2019

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X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

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Validation of Suitable Housekeeping Genes for the Normalization of mRNA Expression for Studying Tumor Acidosis

Cited by 21 publications

References 32 publications

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method

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