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The expression levels of certain genes or proteins may exhibit various features regardless of the tissue type. The housekeeping genes and reference proteins are often used during the normalization of expression studies. Herein, we aim to determine and validate the most stable reference gene and protein for a healthy human fibroblast cell line. This study used a finite fibroblast cell line derived from healthy human skin (CCD1079Sk). The samples between 25 and until senescence occurs at passage 55 were collected. Morphological changes during aging by cultivation were observed in each passage. Seven genes (ACTB, SDHA, GAPDH, RPLP0, VCL, TBP, 18S) were evaluated during cultivation and ranked according to their stability via the RefFinder interface. This study reports strong variations for both gene and protein expression studies and the morphology of the cells. Notably, during cultivation, the cell morphology showed an irreversible change starting from passage 43, before the known replicative lifespan of CCD1079Sk. The lack of an RPLP0 subunit after passage 39 may limit the cultivation range when protein expression is required. GAPDH is also an ideal reference gene and protein when studies include gene and protein expression. Or, as a second option, Vinculin may be the first choice when mRNA expression is required, and β-Actin is the best choice for reference protein when the studies include only protein expression. Plus, such as SDHA, RPLP0, and TBP should be avoided. These results will benefit further studies, including normalization for both gene, protein, and cell type.

Similar to other types of cancer, acidification of tumor microenvironment is an important feature of osteosarcoma, and a major source of cellular stress that triggers cancer aggressiveness, drug resistance, and progression. Among the different effects of low extracellular pH on tumor cells, we have recently found that short-term exposure to acidosis strongly affects gene expression. This alteration might also occur for the most commonly used housekeeping genes (HKG), thereby causing erroneous interpretation of RT-qPCR data. On this basis, by using osteosarcoma cells cultured at different pH values, we aimed to identify the ideal HKG to be considered in studies on tumor-associated acidosis. We verified the stability of 15 commonly used HKG through five algorithms (NormFinder, geNorm, BestKeeper, ΔCT, coefficient of variation) and found that no universal HKG is suitable, since at least four HKG are necessary for proper normalization. Furthermore, according to the acceptable range of values, YWHAZ, GAPDH, GUSB, and 18S rRNA were the most stable reference genes at different pH. Our results will be helpful for future investigations focusing on the effect of altered microenvironment on cancer behavior, particularly on the effectiveness of anticancer therapies in acid conditions.

In nearly every lab, real-time quantitative polymerase chain reaction (qPCR) is used to quantify gene expression. However, a comparison of different samples requires the careful selection of suitable reference genes (RGs), sometimes referred to as housekeeping genes. In the case of vascular smooth muscle cells (vSMCs), it is important to know under which conditions gene expression in isolated and cultured vSMCs can be compared with vSMCs in a healthy blood vessel. We isolated the vSMC-containing layer of the rat aorta (tunica media) and used one (longitudinal) half for direct RNA extraction, while the other half served to isolate and culture vSMCs prior to RNA extraction. First, the expression of the routinely used RGs beta-actin (Actb) and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is investigated in intact media and corresponding cultured vSMCs. Significant differences in their Ct values show that these RGs could not be used for such direct comparisons; therefore, we select 15 different RGs. Only the gene expression of the small ribonuclear protein (snRNP) U2 shows no significant differences between the absolute Ct values of cultured vSMCs and the intact media; moreover, no differences were found between male and female rats in our experimental setup. In conclusion, U2 was shown to be an appropriate (sex-independent) RG to compare relative expression levels of vSMCs in culture to those vSMCs within their physiological tissue environment.

This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.

Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (β-tubulin, cyclophilin, actin, elongation factor 1-α (ef1α), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1α was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

Background A comprehensive analysis of gene expression profiling across tissues can provide necessary information for an in-depth understanding of their biological functions. We performed a large-scale gene expression analysis and generated a high-resolution atlas of the transcriptome in beef cattle. Results Our transcriptome atlas was generated from 135 bovine tissues in adult beef cattle, covering 51 tissue types of major organ systems (e.g., muscular system, digestive system, immune system, reproductive system). Approximately 94.76% of sequencing reads were successfully mapped to the reference genome assembly ARS-UCD1.2. We detected a total of 60,488 transcripts, and 32% of them were not reported before. We identified 2654 housekeeping genes (HKGs) and 477 tissue-specific genes (TSGs) across tissues. Using weighted gene co-expression network analysis, we obtained 24 modules with 237 hub genes (HUBGs). Functional enrichment analysis showed that HKGs mainly maintain the basic biological activities of cells, while TSGs were involved in tissue differentiation and specific physiological processes. HKGs in bovine tissues were more conserved in terms of expression pattern as compared to TSGs and HUBGs among multiple species. Finally, we obtained a subset of tissue-specific differentially expressed genes (DEGs) between beef and dairy cattle and several functional pathways, which may be involved in production and health traits. Conclusions We generated a large-scale gene expression atlas across the major tissues in beef cattle, providing valuable information for enhancing genome assembly and annotation. HKGs, TSGs, and HUBGs further contribute to better understanding the biology and evolution of multiple tissues in cattle. DEGs between beef and dairy cattle also fill in the knowledge gaps about differential transcriptome regulation of bovine tissues underlying economically important traits.

Abstract Although both are salient features of genomes, at first glance ribosomal DNAs and transposable elements are genetic elements with not much in common: whereas ribosomal DNAs are mainly viewed as housekeeping genes that uphold all prime genome functions, transposable elements are generally portrayed as selfish and disruptive. These opposing characteristics are also mirrored in other attributes: organization in tandem (ribosomal DNAs) versus organization in a dispersed manner (transposable elements); evolution in a concerted manner (ribosomal DNAs) versus evolution by diversification (transposable elements); and activity that prolongs genomic stability (ribosomal DNAs) versus activity that shortens it (transposable elements). Re-visiting relevant instances in which ribosomal DNA–transposable element interactions have been reported, we note that both repeat types share at least four structural and functional hallmarks: (1) they are repetitive DNAs that shape genomes in evolutionary timescales, (2) they exchange structural motifs and can enter co-evolution processes, (3) they are tightly controlled genomic stress sensors playing key roles in senescence/aging, and (4) they share common epigenetic marks such as DNA methylation and histone modification. Here, we give an overview of the structural, functional, and evolutionary characteristics of both ribosomal DNAs and transposable elements, discuss their roles and interactions, and highlight trends and future directions as we move forward in understanding ribosomal DNA–transposable element associations.

Quantitative real-time PCR (qPCR) remains a cornerstone method for analyzing gene expression due to its high sensitivity, specificity, and reproducibility. However, for reliable results in relative quantification studies, the choice of an appropriate reference gene is critical to ensure accurate normalization. The expression of commonly used reference genes can vary depending on developmental stage and experimental conditions, making their validation essential. To date, no validated reference genes have been reported for Agrostemma githago L. (corn cockle, Caryophyllaceae). To facilitate research on genes involved in natural product biosynthesis and specialized metabolism regulation, we aimed to identify the most stable reference genes across various plant organs and cultivation conditions of this species. Drawing on previous literature, we have selected seven housekeeping genes widely used for evaluation: actin, β-tubulin, elongation factor 1α, glyceraldehyde-3-phosphate dehydrogenase, histone H3, translation elongation factor 1, and eukaryotic translation initiation factor 5A1 (for which two primer sets were tested). The nucleotide sequences of these potential reference genes were identified from the A. githago transcriptome. Using qRT-PCR, transcript levels of seven potential reference genes were estimated in 40 different A. githago samples, including 25 in vitro samples under various treatment conditions and 15 soil-grown samples representing A. githago organs in different developmental stages. Expression stability of candidate reference genes was assessed using the RefFinder platform, which combines four commonly applied statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ-Ct method. The results revealed that the selection of optimal reference genes varied based on the particular organ, developmental stage and condition being examined. TIF5A1-2 (one of the two primer pairs tested) and GAPHD consistently exhibited the most stable expression under various conditions in vitro. EF1α and H3 exhibited superior performance across different organs of soil-grown plants. Moreover, our integrated analysis enabled the identification of the two most stable, universal reference genes suitable for normalization in A. githago under all tested conditions—H3 and TIF5A1-2. Our work provides a robust foundation for future transcriptomic and functional studies of the specialized metabolism of A. githago and other related species.

Housekeeping genes are commonly used as reference genes for analyzing expression data. We introduce an algorithm that scores and ranks genes based on their expression ubiquity using three criteria: the proportion of samples with high expression, overall expression levels, and expression invariance. The resulting continuous and configurable measure of gene ubiquity overcomes the limitations of current categorical classifications. Using this measure, we systematically evaluate how genes traditionally assumed to be ubiquitously expressed (so-called stable housekeeping genes) respond to drugs. By integrating expression data from the Genotype-Tissue Expression portal (physiological conditions) and the Connectivity Map (drug effects), we identify subsets of genes that maintain strong invariance under chemical stimuli, rendering them particularly suitable as reference genes. At the same time, we show that many seemingly ubiquitous genes are strongly influenced by commonly used drugs. Our findings open up new conceptual perspectives on drug mechanisms and cellular regulatory responses and they call into question the reliability of several classic housekeeping genes. To facilitate broader application of these results, we present an interactive web interface ( https://ubigen.uni-rostock.de ) that allows researchers to explore gene rankings, adjust scoring criteria and analyze specific gene sets. This platform facilitates the selection of robust, ubiquitously expressed reference genes for experimental applications and deepens our understanding of transcriptional regulation under physiological and chemically perturbed conditions.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.