RPS13, a potential universal reference gene for normalisation of gene expression in multiple human normal and cancer tissue samples

Rashid, Mudasir; Shah, Sanket; Natu, Abhiram; Verma, Tripti; Rauniyar, Sukanya; Gera, Poonam; Gupta, Sanjay

doi:10.1007/s11033-021-06828-6

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…We may also speculate that since ribosomal protein synthesis may be increased on the translational level to repair damaged ribosomes in dendrites [ 11 ], temporarily decreased transcription of the corresponding mRNAs is likely not detrimental for neuronal ribosomes and may even be used to redirect transcriptional machinery to activity-induced genes. Notably, RPS13 gene expression was not affected in our experiment; this gene was previously reported to have stable expression in various normal and tumor tissues [ 73 ].…”

Section: Discussionmentioning

confidence: 70%

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin

Beletskiy,

Zolotar,

Fortygina

et al. 2024

Cells

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Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity.

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Section: Discussionmentioning

confidence: 70%

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin

Beletskiy,

Zolotar,

Fortygina

et al. 2024

Cells

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“…Following beta-actin PCR [ 16 ] from all cDNA and control aliquots confirming successful RNA isolation, DNase digestion, and cDNA synthesis, the samples were subjected to MMP1 qPCR. Equine 40S ribosomal protein S13-like (RPS13) served as a reference housekeeping gene in the assay [ 17 , 18 ]. The reactions were conducted in duplicates in final 20 µL volumes containing 10 μL of qPCR TaqMan ® Universal PCR Master Mix (Thermo Fisher Scientific), 1 µL of MMP1 Assay Mix FAM (Assay ID: Ec03468020_m1: Thermo Fisher Scientific) or RPS13 Assay Mix VIC (5′RPS13: 5′-GTGCCCACTTGGCTGAAGTT-3′; 3′RSP13: 5′-TCTTGGCCAGCTTGTAGATCTG-3′; 5′-Probe: VIC-CGTCTGACGACGACGTGAG; Applied Biosystems, Life Technology, Vienna, Austria), 4 µL of sterile water, and 5 µL of template (cDNA or -RT-control, and sterile water as the no-template control).…”

Section: Methodsmentioning

confidence: 99%

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro

Brodesser,

Kummer,

Eichberger

et al. 2024

Cells

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The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial–mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

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“…com/article/10.3390/ijms242015140/s1. References [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67] are cited in the Supplementary Material. Funding: This research was funded by the Spanish Ministry of Science, Innovation, and Universities (Research-Grant: PID2022-1381850B-I00; PID2019-105564RB-I00; Predoctoral contracts: FPU18-06009, PRE2020-094225, FPU18-02485); Junta de Andalucia (BIO-0139); CIBERobn (CIBER is an initiative of Instituto de Salud Carlos III, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain).…”

Section: Institutional Review Board Statementmentioning

confidence: 99%

LRP10, PGK1 and RPLP0: Best Reference Genes in Periprostatic Adipose Tissue under Obesity and Prostate Cancer Conditions

Pérez-Gómez,

Porcel-Pastrana,

De La Luz-Borrero

et al. 2023

IJMS

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Obesity (OB) is a metabolic disorder characterized by adipose tissue dysfunction that has emerged as a health problem of epidemic proportions in recent decades. OB is associated with multiple comorbidities, including some types of cancers. Specifically, prostate cancer (PCa) has been postulated as one of the tumors that could have a causal relationship with OB. Particularly, a specialized adipose tissue (AT) depot known as periprostatic adipose tissue (PPAT) has gained increasing attention over the last few years as it could be a key player in the pathophysiological interaction between PCa and OB. However, to date, no studies have defined the most appropriate internal reference genes (IRGs) to be used in gene expression studies in this AT depot. In this work, two independent cohorts of PPAT samples (n = 20/n = 48) were used to assess the validity of a battery of 15 literature-selected IRGs using two widely used techniques (reverse transcription quantitative PCR [RT-qPCR] and microfluidic-based qPCR array). For this purpose, ΔCt method, GeNorm (v3.5), BestKeeper (v1.0), NormFinder (v.20.0), and RefFinder software were employed to assess the overall trends of our analyses. LRP10, PGK1, and RPLP0 were identified as the best IRGs to be used for gene expression studies in human PPATs, specifically when considering PCa and OB conditions.

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RPS13, a potential universal reference gene for normalisation of gene expression in multiple human normal and cancer tissue samples

Cited by 5 publications

References 31 publications

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro

LRP10, PGK1 and RPLP0: Best Reference Genes in Periprostatic Adipose Tissue under Obesity and Prostate Cancer Conditions

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