Quantitative Gene Expression Analysis Research Articles

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses.

Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress conditions like drought, salt, heat and cold stress adversely affect the productivity and quality of mulberry leaves. Quantitative real time PCR (qPCR) is a reliable and widely used method to identify abiotic stress responsive genes and molecular mechanism in different plant species. Selection of suitable reference genes is important requirement for normalizing the expression of genes through qRT-PCR study. In the present study, we have selected eight candidate reference genes in mulberry for analyzing their expression stability in different abiotic stress treatments including drought, salt, heat and cold stresses. The expression stability of these reference genes was determined using geNorm, NormFinder and RefFinder statistical algorithms. The results showed that Ubiquitin and protein phosphatase 2A regulatory subunit A (PP2A) were the most stable genes across all the treatment samples. However, analysis of individual stresses revealed different expression profiles and stability of reference genes. Actin3 and PP2A were most stable in drought and salt conditions respectively. RPL3 most preferred in heat stress and Ubiquitin was most stable in cold stress. We propose the ubiquitin and PP2A are the preferred reference genes for normalization of gene expression data from abiotic stresses. In addition, Actin3 are preferred for drought stress, PP2A for salt stress, RPL3 for heat stress and Ubiquitin for cold stress studies.

Gene Expressions Analysis of Seed Physiological Quality in Soybean Cultivars

Soybean seed production with high physiological quality is important for ensuring populations of recommended plants for each cultivar. It is also known that there is genetic variability for this characteristic among the soybean cultivars. The objective was to study the genes expressionof protein and gene transcript involved in the physiological quality of soybean seeds. The seeds of CD 206 and CD 201 cultivars were classified as high quality while Savana and Emgopa 316 cultivars were classified as low quality by germination and vigor tests. Physiological, enzymatic and transcripts analysis were held in seeds of each cultivar during two harvest seasons, R8 and R8 + 15 days; at protein level the alcohol dehydrogenase isoenzyme systems (ADH), phosphoglucoisomerase (PGI), superoxide dismutase (SOD), catalase (CAT), malate dehydrogenase (MDH) and isocitrate lyase (ICL). The PCR technique in real time (qRT-PCR) was used for transcritome studies for quantitative gene expression analysis. The method used was the comparative Ct considering the relative expression levels in relation to cultivar of high quality CD 201. It is concluded that there is higher enzymes expression involved in respiration, alcohol dehydrogenase (ADH), malate dehydrogenase (MDH) and phosphoglucoisomerase (PGI), in seeds with high physiological quality, especially when harvesting is delayed. The proteomic and transcriptomic profiles related to the genes involved in free radicals removing systems in soybeans varies among cultivars and the season of seed harvest.

Quantitative Analysis of Gene Expression During Calcium Homeostasis in E. coli

Despite a wealth of knowledge regarding calcium’s role as secondary messenger in eukaryotic cells, relatively little is known about calcium homeostasis in bacteria. Our study employing molecular genetics and transcriptome analysis of calcium homeostasis previously exhibited a calcium regulation of gene expression in elevated or depleted calcium throughout E. coli genome. Here we report the results of our subsequent investigation into quantitative analysis using quantitative reverse transcriptase PCR (qRT-PCR) of gene expression in subsets of genes from wild-type and mutant strains with addition of calcium and chelation of calcium. Data from qRT-PCR reveal very high upregulation of genes yjeE and secA indicating their possible role as overactive calcium efflux systems in E. coli. Calcium is highly likely to be involved in stress response as evidenced by upregulation of marB in transposon mutants and by 10-fold induction of rpoS in elevated calcium condition in mutants defective in calcium transport.
 Bangladesh J Microbiol, Volume 34 Number 2 December 2017, pp 47-54

Quantification of Cauline Leaf Abscission in Response to Plant Pathogens.

Abscission is a process that allows plants to shed unwanted organs. Plants can use abscission as a defense mechanism to shed leaves that are heavily infected with pathogenic bacteria. By shedding infected leaves, plants completely eliminate the bacteria from the plant body, thus preventing further spreading of the disease. A lot is known about how plants limit the growth of pathogenic bacteria in vegetative leaf tissues. Much less is known about how plants defend themselves in non-vegetative developmental stages and how they use organ level responses such as leaf abscission for defense. Organ level defense responses can be effectively studied in the Pseudomonas syringae-triggered leaf abscission system in Arabidopsis. This method article describes detailed procedures for quantitative analysis of cauline leaf abscission including dissecting abscission zones for extraction of RNA and proteins for quantitative gene or protein expression analysis. The method described for molecular analysis of abscission zones could also be used in other cases where tissue is extremely limiting.

Transcriptomics reveals complex kinetics of dorsal-ventral patterning gene expression in the mandibular arch.

The mandibular or first pharyngeal arch forms the upper and lower jaws in all gnathostomes. A gene regulatory network that defines ventral, intermediate, and dorsal domains along the dorsal-ventral (D-V) axis of the arch has emerged from studies in zebrafish and mice, but the temporal dynamics of this process remain unclear. To define cell fate trajectories in the arches we have performed quantitative gene expression analyses of D-V patterning genes in pharyngeal arch primordia in zebrafish and mice. Using NanoString technology to measure transcript numbers per cell directly we show that, in many cases, genes expressed in similar D-V domains and induced by similar signals vary dramatically in their temporal profiles. This suggests that cellular responses to D-V patterning signals are likely shaped by the baseline kinetics of target gene expression. Furthermore, similarities in the temporal dynamics of genes that occupy distinct pathways suggest novel shared modes of regulation. Incorporating these gene expression kinetics into our computational models for the mandibular arch improves the accuracy of patterning, and facilitates temporal comparisons between species. These data suggest that the magnitude and timing of target gene expression help diversify responses to patterning signals during craniofacial development.

Antidiabetic Potential of Green Seaweed Enteromorpha prolifera Flavonoids Regulating Insulin Signaling Pathway and Gut Microbiota in Type 2 Diabetic Mice.

This study aimed to investigate the antidiabetic activity of water-ethanol extract of green macroalgae Enteromorpha prolifera (EPW) and its flavonoid-rich fraction less than 3 kDa (EPW3) in type 2 diabetic mice induced by streptozotocin and a high-sucrose/high-fat diet. The major active compounds were identified using ultra-performance liquid chromatography coupled with quadrupole-time-of-flight-tandem mass spectrometry. Quantitative gene expression analysis of the insulin signaling pathway was performed. The effects of EPW3 on gut microflora in diabetic mice were analyzed by high-throughput 16S rRNA gene sequencing. The results showed EPW3 treatment decreased the fasting blood glucose, improved oral glucose tolerance, and protected against liver and kidney injury with reduced inflammation in diabetic mice. The active principle of EPW3 revealed hypoglycemiceffect as indicated by activation of the IRS1/PI3K/AKT and inhibition of the JNK1/2 insulin pathway in liver. Furthermore, the treatment significantly enriched the abundance of Lachnospiraceae and Alisties, which were positive correlation of metabolic phenotypes. These findings indicated that EPW3 possessed great therapeutic potential as adjuvant therapy for type 2 diabetes.

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression in Hainan medaka (Oryzias curvinotus)

Quantitative real-time PCR (qRT-PCR) is a powerful and sensitive methodology to analyze expression of target genes. qRT-PCRs rely on selecting suitable reference gene. In the present study, 17 candidate reference genes in Hainan medaka, Oryzias curvinotus, were selected, and candidate genes were studied for mRNA expression levels and stabilities in different tissues and at different stages of embryonic development. Expression patterns were analyzed using RefFinder and geNorm to test for suitability of the gene as a reference gene. RefFinder analysis ranked the stability of the reference genes in the following order (Top 5): rps4x > eef1b > 60srp > dcakd > eef1a in different tissues, and mrp13 > rps20 > actb > sbds > adp in embryonic development. Similarly, geNorm selected eef1b and rps4x as the two optimal reference genes for normalizing target genes expression in Hainan medaka. The results demonstrated that the expression of reference genes is variable under different experimental conditions, highlighting the importance of validating the stability and optimal number of reference genes before doing the qRT-PCR experiment.

2021. An Ultra-Rapid Host Response Assay to Discriminate Between Bacterial and Viral Infections Using Quantitative Isothermal Gene Expression Analysis

BackgroundAccurate diagnosis and treatment of bacterial infection is critical for improving patient outcomes. However, over-prescription of antibiotics has contributed to C. difficile-infections and to the emergence of antimicrobial resistance. As assessing bacterial infection by culture is slow and molecular pathogen detection is limited in scope, an unmet need remains for a rapid test to differentiate between viral and bacterial infections. We have previously identified a set of 7 host response biomarkers demonstrating an AUROC of 0.91–0.93 for separating bacterial from viral infections across multiple independent cohorts. A clinical in-vitro diagnostic test (IVD) using these markers must be very fast to integrate with physician workflows. Loop-mediated isothermal amplification (LAMP) represents a rapid amplification solution with the potential to meet these needs. We describe an ultra-rapid LAMP assay designed to quantitate these markers for applications in point-of-care decision making.MethodsLAMP primers for gene expression analysis of selected markers with a housekeeping control were designed for mRNA specificity by targeting FIP primers to splice junctions. Assay specificity, sensitivity and linear dynamic range were evaluated using serial dilution of control material. RNA extracted from preserved patient samples was evaluated by LAMP for concordance with NanoString® nCounter® data (nCounter).ResultsIterative optimization of primer design resulted in RT-LAMP assays that selectively amplify target mRNA. Assays demonstrate a linear dynamic range spanning 6 orders of magnitude and a quantitative LOD of about 103 copies. Quantitation of relative expression levels showed good concordance with nCounter data in 10 healthy, 6 viral and 6 bacterial patient samples, with average threshold times of <15 minutes.ConclusionAccurate discrimination of bacterial and viral infection can be achieved on a true point-of-care timescale using our LAMP strategy. This assay could be run on a standard thermal cycler, or any quantitative molecular instrument that allows to measure at least eight targets. An IVD test distinguishing between bacterial and viral infections could aid in antimicrobial treatment decisions and thereby minimize over-prescription of antibiotics.Disclosures D. Rawling, Inflammatix Inc.: Employee, Salary. W. Nie, Inflammatix Inc.: Employee, Salary. M. Remmel, Inflammatix Inc.: Employee, Salary. M. Eshoo, Inflammatix Inc.: Employee, Salary. J. Romanowsky, Inflammatix Inc.: Employee, Salary. O. Liesenfeld, Inflammatix Inc.: Employee, Salary. T. Sweeney, Inflammatix Inc.: Employee, Salary.

Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.

Different Genetic Expression Profiles of Oxidative Stress and Apoptosis-Related Genes in Crohn’s Disease

Background/Aims: Increased oxidative stress and decreased immune cell apoptosis have been reported to be important factors in the pathogenesis of Crohn’s disease (CD). Our aim was to characterize the genetic expression of molecules implicated in the regulation of oxidative stress and apoptosis in peripheral white mononuclear cells of 18 healthy volunteers (controls) and 20 patients at the onset of CD (active CD [aCD]): 10 who achieved remission (inactive CD [iCD]) and 10 who did not present a complete and deep response to treatment (aCD-T). Methods: mRNA expression was measured by the Agena MassARRAY quantitative gene expression analysis application. The genes analyzed were Fas-receptor (FASR), Fas-ligand (FASL), signal transducer and activator of transcription 1 (STAT1), nuclear factor kappa-light-chain-­enhancer of activated B cells (NFKB1), apoptosis signal-regulating kinase 1 (ASK1), serine/threonine-protein kinase H1 (PSKH1), ATP-binding cassette sub-family B1 (ABCB1) and peptidylprolyl isomerase D (PPID). Results: During a CD flare, we found specific upregulated expression of the genes STAT1 and PSKH1, whereas ABCB1 and FASL were downregulated. In the patients with iCD, FASR and NFKB1 were upregulated. The expression levels of NFKB1, STAT1 and ABCB1 did not show any difference in patients with aCD at the onset of the disease and after treatment (aCD-T). The expression levels of PPID and ASK1 did not show any differences in the patients with aCD, iCD and the controls. We have also reviewed the cellular function and role of these genes in CD. Conclusions: These findings contribute to improving the understanding of the pathogenesis of CD and highlight potential genes involved.

RNA-seq: Basic Bioinformatics Analysis.

Quantitative analysis of gene expression is crucial for understanding the molecular mechanisms underlying genome regulation. RNA-seq is a powerful platform for comprehensive investigation of the transcriptome. In this unit, we present a general bioinformatics workflow for the quantitative analysis of RNA-seq data and describe a few current publicly available computational tools applicable at various steps of this workflow. These tools comprise a pipeline for quality assessment and quantitation of RNA-seq data that starts from raw sequencing files and is focused on the identification and analysis of genes that are differentially expressed between biological conditions. © 2018 by John Wiley & Sons, Inc.

A High-throughput Quantitative Expression Analysis of Cancer-related Genes in Human HepG2 Cells in Response to Limonene, a Potential Anticancer Agent.

Citrus bioactive compounds, as active anticancer agents, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of the pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. Highthroughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene- driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from the most to the least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines.

Increased inflammatory responsiveness of peripheral blood mononuclear cells (PBMCs) to in vitro NOD2 ligand stimulation in patients with ankylosing spondylitis

Background: Ankylosing spondylitis (AS) is a common debilitating rheumatic disease in which the innate immune components especially the Interleukin (IL)-23/IL-17 axis related genes play important role in its pathogenesis. Nucleotide binding oligomerization domain-containing protein (NOD)2, as an innate receptor, is critical for IL-23 production in cells. Therefore, we aimed to stimulate NOD2 signaling and study its effects on cytokine production in peripheral blood mononuclear cells (PBMC) of these patients.Methods: PBMCs from 18 patients with active AS and 18 healthy individuals were separated by Ficoll-Hypaque density gradient centrifugation and cultured in the presence of muramyl dipeptide (MDP), as NOD2 ligand. Quantitative expression analysis of NOD1, NOD2, RIPK2, SLC15A4, NLRP1, NLRP3, IL23A, IL17A, IL1B, and TNFA genes was performed using Real-time polymerase chain reaction (PCR). Finally, protein changes of IL23A and IL17A expression were validated using enzyme linked immunosorbent assay (ELISA).Results: Apart from NOD1 that tend to be downregulated in the controls, all the selected genes showed overexpression in response to MDP in cells from the studied groups. Except RIPK2, all the genes had higher expression changes upon MDP stimulation in the AS population. Overexpression of IL23A and IL17A were confirmed at protein levels using ELISA. The strong positive correlation between NLRP3 and NOD2 was decreased after stimulation but new correlations between NLRP3 and IL1B, RIPK2 and SLC15A4 were observed after treatment.Conclusions: This study indicated that AS PBMCs were hyper-responsive to MDP stimulation. This observation implies an important role of NOD2 in the pathogenesis of inflammatory diseases including AS.

Effectof Castration on Some Immunological Aspects in Male Albino Rat

The current study aimed to evaluate the effect of surgical and chemical castration on some immunological aspects in adult male albino rats. A total number of forty adult male albino rats weighing 220±20 g and averaging 4 months old were used. Rats were divided into four equal groups. Group 1, intact control group; group 2, sham castrated group; group3, surgically castrated group and group 4, chemically castrated group. Relative thymus and spleen weight, peripheral total and differential leukocytic count and relative expression of TNFα gene were recorded at 2 and 4 weeks after castration. The obtained results revealed that surgical and chemical castration resulted in a significant increase in relative thymus and spleen weight. Peripheral total leukocytic count was significantly increased in surgically and chemically castrated groups as compared to intact control and sham groups. Differential leukocytic count revealed a significant increase in lymphocyte number and a significant decrease in neutrophil number in surgically and chemically castrated groups as compared to intact control and sham groups. Real-time quantitative PCR analysis of TNFα gene expression in spleen revealed that surgical and chemical castration resulted in significant up regulation of TNFα gene expression. So, it concluded that surgical and chemical castration improved immunity and testosterone has an immunosuppressive effect in adult male albino rats.

Comprehensive analysis of wintersweet flower reveals key structural genes involved in flavonoid biosynthetic pathway

Wintersweet (Chimonanthus praecox (L.)), with an over-one-thousand-years long history in cultivation, is still a popular ornamental woody plant in China. The tepals of wintersweet flower are waxy in nature and the overall color of the flower is yellow, while the inner tepals range from yellow to red, which makes it an ideal plant to study floral color formation in ornamental shrubs. In our current work, HPLC analysis revealed that the principal pigments in tepals were the metabolite of flavonoids. All the tepals were containing quercetin, kaempferol 3‑O‑rutinoside and rutin while cyanidin‑3‑O‑glucoside and cyanidin‑3‑O‑rutinoside were only found in the in the red tepals. Moreover, we found the rutin as the principal component of all the pigments revealed. As well as in this study, a reference transcriptome library constructed from two varieties H29 and H64 flower. Further, 30 proteins of flavonoid biosynthesis pathway were identified in H29 flower using proteome analysis. Based on these dataset, the flavonoid biosynthesis pathway was also speculated. After quantitative analysis of gene expression, we found that ANS act as an on-off switch for the accumulation of red pigments and had positive correlations with various steps genes of the flavonoid pathway. This expression profiling demonstrates that no gene products compete for common substrates to redirect the metabolic flux in wintersweet. It is also demonstrated that high expression of F3′H would provide sufficient content of the precursor, dihydroquercetin, for both flavonol and anthocyanin biosynthesis. The results help us to deepen and enrich the gene resource of color formation in wintersweet flower, and provide specific breeding strategies for increasing diversity of flower color.

2D DIGE proteomic analysis reveals fasting-induced protein remodeling through organ-specific transcription factor(s) in mice.

Overnight fasting is a routine procedure before surgery in clinical settings. Intermittent fasting is the most common diet/fitness trend implemented for weight loss and the treatment of lifestyle‐related diseases. In either setting, the effects not directly related to parameters of interest, either beneficial or harmful, are often ignored. We previously demonstrated differential activation of cellular adaptive responses in 13 atrophied/nonatrophied organs of fasted mice by quantitative PCR analysis of gene expression. Here, we investigated 2‐day fasting‐induced protein remodeling in six major mouse organs (liver, kidney, thymus, spleen, brain, and testis) using two‐dimensional difference gel electrophoresis (2D DIGE) proteomics as an alternative means to examine systemic adaptive responses. Quantitative analysis of protein expression followed by protein identification using matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry (MALDI‐TOFMS) revealed that the expression levels of 72, 26, and 14 proteins were significantly up‐ or downregulated in the highly atrophied liver, thymus, and spleen, respectively, and the expression levels of 32 proteins were up‐ or downregulated in the mildly atrophied kidney. Conversely, there were no significant protein expression changes in the nonatrophied organs, brain and testis. Upstream regulator analysis highlighted transcriptional regulation by peroxisome proliferator‐activated receptor alpha (PPARα) in the liver and kidney and by tumor protein/suppressor p53 (TP53) in the thymus, spleen, and liver. These results imply of the existence of both common and distinct adaptive responses between major mouse organs, which involve transcriptional regulation of specific protein expression upon short‐term fasting. Our data may be valuable in understanding systemic transcriptional regulation upon fasting in experimental animals.

Epithelial-to-mesenchymal transition–related transcription factors are up-regulated in ependymomas and correlate with a poor prognosis

Epithelial-to-mesenchymal transition (EMT) plays an important role in invasion and metastasis of various cancers including gliomas. EMT has also been linked to cancer stem cells and resistance to chemotherapy. An initial in-silico data mining in a published ependymoma (EPN) patient series (GSE21687) revealed up-regulation of EMT transcription factors in tumor samples. Furthermore, quantitative real-time polymerase chain reaction-based gene expression analysis of EMT transcription factors in 96 EPNs showed significant up-regulation of SNAI1, SNAI2, ZEB1, and TWIST1 as compared with normal brain, associated with up-regulation of CDH2/N-cadherin and down-regulation of CDH1/E-cadherin. Although this was observed in varying degrees in all clinicopathological-molecular subgroups of EPNs, it was most evident in supratentorial EPNs harboring fusions of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) gene and in posterior fossa EPNs. Immunohistochemistry performed in 60 of the above cases corroborated with gene expression patterns, and immunopositivity for Snail, Slug, Zeb1, and Twist1 was observed in 80%, 80%, 81%, and 63% of all EPNs. Immunopositivity for N-cadherin and E-cadherin was observed in 76.6% and 2% of the cases, respectively. Univariate Cox regression analysis showed that low expression of CDH1/E-cadherin (P = .002) and high expression levels of CDH2/N-cadherin (P < .001), SNAI1/Snail (P = .023), SNAI2/Slug (P < .001), and ZEB1 (P < .001) were associated with shorter progression-free survival. Here, we report for the first time the existence of EMT-like phenotype in EPNs. These factors could represent new prognostic and therapeutic targets in EPN.

Quality Assessment of Surgical Disc Samples Discriminates Human Annulus Fibrosus and Nucleus Pulposus on Tissue and Molecular Level.

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.

Molecular Phenotyping of Telomerized Human Bone Marrow Skeletal Stem Cells Reveals a Genetic Program of Enhanced Proliferation and Maintenance of Differentiation Responses.

ABSTRACTLong‐term in vitro expansion of bone marrow stromal (skeletal) stem cells (also known as human mesenchymal stem cells [hMSC]) is associated with replicative senescence and impaired functions. We have previously reported that telomerization of hMSC through hTERT overexpression led to bypassing a replicative senescence phenotype and improved in vitro and in vivo functions. However, the molecular consequence of telomerization is poorly characterized. Thus, we compared the molecular phenotype of a well‐studied telomerized hMSC (hMSC‐TERT) cell line with primary hMSC. At a cellular level, both cell populations exhibited strong concordance for the known hMSC CD markers, similar responses to osteoblast (OB) differentiation induction, and formed heterotopic bone in vivo. Overall gene expression was highly correlated between both cell types with an average Pearson's correlation coefficient (R2) between the gene expression of all primary hMSC and all hMSC‐TERT samples of 0.95 (range 0.93–0.96). Quantitative analysis of gene expression of CD markers, OB cell markers, and transcription factors (TF) showed a high degree of similarity between the two cell populations (72%, 77%, and 81%, respectively). The hMSC‐TERT population was enriched mainly for genes associated with cell cycle and cell cycle signaling when compared with primary hMSC. Other enrichment was observed for genes involved in cell adhesion and skeletal system development and immune response pathways. Interestingly, hMSC‐TERT shared a telomerization signature with upregulation of cancer/testis antigens, MAGE, and PAGE genes. Our data demonstrate that the enhanced biological characteristics of hMSC after telomerization are mainly due to enhanced expression of cell proliferation genes, whereas gene expression responses to differentiation are maintained. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research

Housekeeping Gene Stability in Human Mesenchymal Stem and Tendon Cells Exposed to Tenogenic Factors.

The use of biochemical inducers of mesenchymal stem cell (MSC) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. Bone morphogenetic protein 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes. To date, no published studies have evaluated the stability of housekeeping genes in MSCs during tenogenic induction. Three candidate housekeeping genes (YWHAZ, RPL13A, and GAPDH) in human MSCs from bone marrow (BMSCs), adipose tissue (ASCs), and tendon cells (TCs) supplemented with BMP-12 or AA and bFGF in comparison with control untreated cells for 3 and 10 days were evaluated. GeNorm, NormFinder, and BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability and the overall ranking was determined by using by the RefFinder algorithm. In all culture conditions, YWHAZ was the most stable gene and RPL13A was the second choice. YWHAZ and RPL13A were the two most stable genes also for ASCs and BMSCs, regardless of the time point analyzed, and for TCs at 10 days of tenogenic induction. Only for TCs at 3 days of tenogenic induction were GAPDH and YWHAZ the best performers. In conclusion, our findings will be useful for the proper selection of housekeeping genes in studies involving MSCs cultured in the presence of tenogenic factors, to obtain accurate and high-quality data from quantitative gene expression analysis.