RT-qPCR Gene Expression Research Articles

Selection and validation of reference genes for the normalization of RT-qPCR gene expression data in Rheum tanguticum (Polygonaceae) under various abiotic stresses.

Rheum tanguticum, an endemic species from the Qinghai-Xizang Plateau, is a significant perennial and medicinal plant recognized for its robust resistance to abiotic stresses, including drought, cold, and salinity. To advance the understanding of stress-response mechanisms in R. tanguticum, this study aimed to establish a reliable set of housekeeping genes as references for normalizing RT-qPCR gene expression analyses. Ten candidate genes were selected based on prior studies of related species and transcriptomic data for R. tanguticum. Their expression stability was evaluated across three tissue types (leaf, stem, and root) under four abiotic stress treatments using a comprehensive suite of bioinformatics tools. Our findings revealed that RtEF1α was the most stable reference gene in salt- and drought-stressed roots and drought-stressed stems, while RtTUB was most stable in salt-stressed stems. RtSAND was identified as the most stable reference gene in salt-, drought-, heavy metal-, and hormone-stressed leaves. For heavy metal- and hormone-stressed roots and heavy metal-stressed stems, RtUBC demonstrated the highest stability, whereas RteIF6A was most stable in hormone-stressed stems. Furthermore, the utility of these reference genes was validated by assessing the expression pattern of the drought-related gene RtNAC. This study is the first report on selecting and validating reference genes of R. tanguticum under various stress conditions, which will benefit future investigations of the genomic mechanisms involved on stress resistance in this species.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Green Synthesis of a Novel Phytoalexin Derivative: In Silico Profiling, Apoptotic Induction, and Antiproliferative Activity against MCF-7 cells - From Vineyards to Potent Anticancer Drug Molecule.

Resveratrol's structural similarity to commercialized anti-breast cancer medications such as Tamoxifen underlines its potential as a promising option for developing successful anti-breast cancer drugs. However, the pharmacokinetic issues associated with resveratrol, such as its low bioavailability, have piqued the attention of researchers in developing novel derivatives. A novel phytoalexin derivative, RsvD1, was successfully synthesized using resveratrol extracted from green grape peels as a precursor to investigate its anti-breast cancer efficacy on Estrogen receptor (ER) positive and negative breast cancer cells. The comparative analysis revealed that RsvD1 exhibited remarkable radical scavenging ability (IC50 = 2.21 μg/mL), surpassing the control, Trolox (IC50 = 6.3 μg/mL). Furthermore, RsvD1 demonstrated enhanced and selective antiproliferative activity against ER-positive MCF-7 cells (IC50 = 20.09 μg/mL) compared to resveratrol, the parent molecule (IC50 = 30.90 μg/mL). Further investigations unveiled that RsvD1 induced apoptosis and DNA damage in MCF-7 cells, leading to cell cycle arrest at the G0/G1 phase after 24 hours of incubation. RTqPCR gene expression analysis indicated that RsvD1 down-regulated the CAXII (ER-dependent) genes. In silico predictions demonstrated that RsvD1 possesses promising potential as a drug candidate due to its drug-like characteristics and favourable ADMET profile. Moreover, molecular docking studies provided insights into the theoretical binding mode between RsvD1 and ERα protein. The study highlights the therapeutic potential of the synthesized resveratrol derivative, RsvD1, positioning it as a promising scaffold for developing novel analogues with improved therapeutic properties and selectivity, specifically targeting ER+ breast cancer cells. Moreover, the compound's non-cytotoxic yet antiproliferative properties, coupled with its capability to induce programmed cell death and cell cycle arrest, enhance its potential as a highly effective drug candidate. As a result, this paves a promising path for the development of innovative and selective inhibitors targeting ER+ breast cancer with enhanced efficacy.

Compensative Resistance to Erastin-Induced Ferroptosis in GPX4 Knock-Out Mutants in HCT116 Cell Lines.

Ferroptosis results from the accumulation of oxidized and damaged lipids which then leads to programmed cell death. This programmed process is iron-dependent, and as a fundamental biological process, plays a crucial role in tissue homeostasis. The ferroptosis molecular pathway depends on self-regulatory genes: GPX4; TFRC; ACSL4; FSP1; SLC7A11, and PROM2. Some of them were considered here as ferro-sensitive or ferro-resistance markers. We examined the impact of GPX4 gene knock-out, using the CRISPR/Cas-9 technique, on ferroptosis induction in the HCT116 colorectal cancer cell line. The results confirmed that cells lacking the GPX4 gene (GPX4 KO) should be more susceptible to ferroptosis after erastin treatment. However, the decrease in cell viability was not as significant as we initially assumed. Based on the lipid peroxidation markers profile and RT-qPCR gene expression analysis, we revealed the activation of an alternative antioxidant system supporting GPX4 KO cells, mostly for cellular ferroptotic death avoidance. Increased expression of FSP1 and PRDX1 genes in knock-out mutants was associated with their function-recognized here as ferroptosis suppressors. For such reasons, studies on the role of GPX4 and other crucial genes from the ferroptotic pathway should be explored. Despite promising prospects, the utilization of ferroptosis mechanisms in cancer therapy remains at the stage of experimental and in vitro preclinical studies.

Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi

Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease, but its fatality rate is high (96%) mainly because of delay in initiation of treatment due to misdiagnosis and lack of a specific treatment. The analysis of gene expression by quantitative real-time PCR in N. gruberi could be a highly efficient means to understand the pathogenicity of N. fowleri and also to find drug targets. Accurate RT-qPCR analysis requires correct normalization of gene expression data using reference genes (RG), whose expression should be constant under different experimental conditions. In this study, six genes, representing the most frequently used housekeeping genes, were selected for evaluation as reference genes in N. gruberi. The expression and stability of these genes was evaluated employing four algorithms (geNorm, NormFinder, BestKeeper and RefFinder). This work shows significant variations of the stability of RGs depending on the algorithms employed and on the experimental conditions (i.e. logarithmic, stationary, heat-shock and oxidative stress). The geNorm, NormFinder and RefFinder analysis of all the experimental conditions in combination revealed that ACT and G6PD were the most stable RGs. While BestKeeper analysis showed that 18S and TBP were the most stable RGs. Moreover, normalization of HSP90 gene expression with the most stable RGs resulted in an upregulation whereas when the normalization was done with the unstable RGs, the gene expression was not reliable. Hence, the implications of this study are relevant to gene expression studies in N. gruberi.

Incorporating Primer Amplification Efficiencies in Quantitative Reverse Transcription Polymerase Chain Reaction Experiments; Considerations for Differential Gene Expression Analyses in Zebrafish.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is commonly used to measure the mRNA expression of target genes in zebrafish. Gene expression values from RT-qPCR are typically reported as relative fold-changes, and relative quantification of RT-qPCR data incorporates primer amplification efficiency values for each target gene. We describe the influence of the primer amplification efficiency analysis method on RT-qPCR gene expression fold-change calculations. This report describes (1) a sample analysis demonstrating incorporation of primer amplification efficiency into RT-qPCR analysis for comparing gene expression of a gene of interest between two groups when normalized to multiple reference genes, (2) the influence of differences in primer amplification efficiencies between measured genes on gene expression differences calculated from theoretical delta-Cq (dCq) values, and (3) an empirical comparison of the influence of three methods of defining primer amplification efficiency in gene expression analyses (delta-delta-Cq [ddCq], standard curve, LinRegPCR) using mRNA measurements of a set of genes in zebrafish embryonic development. Given the need to account for the influence of primer amplification efficiency along with the simplicity of using software programs (LinRegPCR) to measure primer amplification efficiency from RT-qPCR data, we encourage using empirical measurements of primer amplification efficiency for RT-qPCR analysis of differential gene expression in zebrafish.

Selection and validation of internal control genes for quantitative real-time RT‒qPCR normalization of Phlebopus portentosus gene expression under different conditions.

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT‒qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Centipedegrass under Different Abiotic Stress.

As a C4 warm-season turfgrass, centipedegrass (Eremochloa ophiuroides (Munro) Hack.) is known for its exceptional resilience to intensive maintenance practices. In this research, the most stably expressed reference genes in the leaves of centipedegrass under different stress treatments, including salt, cold, drought, aluminum (Al), and herbicide, were screened by the quantitative real-time PCR (RT-qPCR) technique. The stability of 13 candidate reference genes was evaluated by software GeNorm V3.4, NormFinder V20, BestKeeper V1.0, and ReFinder V1.0. The results of this experiment demonstrated that the expression of the UBC (ubiquitin-conjugating enzyme) remained the most stable under cold and Al stress conditions. On the other hand, the MD (malate dehydrogenase) gene exhibited the best performance in leaf tissues subjected to salt and drought stresses. Under herbicide stress, the expression level of the RIP (60S ribosomal protein L2) gene ranked the highest. The expression levels of abiotic stress-associated genes such as PIP1, PAL, COR413, ALMT9, and BAR were assessed to validate the reliability of the selected reference genes. This study provides valuable information and reference points for gene expression under abiotic stress conditions in centipedegrass.

Comparative Analyses of Superoxide Dismutase (SOD) Gene Family and Expression Profiling under Multiple Abiotic Stresses in Water Lilies

Plants in their natural habitat frequently face different biotic and abiotic stresses, which lead to the production of reactive oxygen species (ROS) that can damage cell membranes, cause peroxidation and deterioration of macromolecules, and ultimately result in cell death. Superoxide dismutase (SOD), a class of metalloenzymes, is primarily found in living organisms and serves as the principal line of defense against ROS. The SOD gene family has not yet been characterized in any species of water lily from the genus Nymphaea. The present study aims to conduct a genome-wide study to discover SOD genes in four representative water lily species. In our present comparative study, we discovered 43 SOD genes in the genomes of four water lily species. The phylogenetic investigation results revealed that SOD genes from water lily and closely related plant species formed two distinct groups, as determined by their binding domains with high bootstrap values. Enzymatic ion-binding classified the SOD gene family into three groups, FeSOD, Cu/ZnSOD, and MnSOD. The analysis of gene structure indicated that the SOD gene family exhibited a relatively conserved organization of exons and introns, as well as motif configuration. Moreover, we discovered that the promoters of water lily SODs contained five phytohormones, four stress-responsive elements, and numerous light-responsive cis-elements. The predicted 3D protein structures revealed water lily SODs form conserved protein dimer signatures that were comparable to each other. Finally, the RT-qPCR gene expression analysis of nine NcSOD genes revealed their responsiveness to heat, saline, cold, cadmium chloride, and copper sulphate stress. These findings establish a basis for further investigation into the role of the SOD gene family in Nymphaea colorata and offer potential avenues for genetic enhancement of water lily aquaculture.

Selection and validation of reference genes for RT-qPCR gene expression studies in Candida viswanathii cultivated under different grown conditions

The properties presented by Candida viswanathii's lipases turn this specie into a promising producer of potentially applicable lipases in several industrial sectors, such as: food, textiles, in the oleochemical and paper industries, and also in different pharmaceutical applications. However, studies for elucidating growth and developmental processes at the molecular level in this species are still incipient. Performing such kinds of studies often rely on the use of the RT-qPCR, which is a highly sensitivity technique, but whose parameters must be carefully planned for achieving reliable data. Among the crucial parameters required for achieving reliable results through this technique, the use of appropriated and validated reference genes is one the most important, constituting a bottleneck, mainly in species where molecular studies are scarce. Thus, the aim of this study was to determine the best reference genes for RT-qPCR gene expression studies in C. viswanathii grown in culture media containing four different carbon sources (Olive oil, Triolein, Tributyrin, and Glucose). Eleven candidate reference genes (ACT, GPH1, AGL9, RPB2, SAP1, PGK1, TAF10, UBC13, TFC1, UBP6, and FBA1) were analyzed for their expression patterns and stability. Analysis of gene expression stability was performed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms, and validation of the results was performed through analyzing the expression of a lipase gene, CvLIP4. Analyzing the four treatments together, CvACT and CvRPB2 constituted the best reference gene pair. When treatments are analyzed individually, CvRPB2/CvACT, CvFBA1/CvAGL9, CvPGK1/CvAGL9 and CvACT/CvRPB2 were the best reference gene pairs for the culture media containing olive oil, triolein, tributyrin, and glucose as carbon sources, respectively. These results are essential and form the basis for the development of relative gene expression studies in C. viswanathii, since adequate reference genes are crucial for the reliability of RT-qPCR data.

Correlating mechanical and gene expression data on the single cell level to investigate metastatic phenotypes

Stiffness has been observed to decrease for many cancer cell types as their metastatic potential increases. Although cell mechanics and metastatic potential are related, the underlying molecular factors associated with these phenotypes remain unknown. Therefore, we have developed a workflow to measure the mechanical properties and gene expression of single cells that is used to generate large linked-datasets. The process combines atomic force microscopy to measure the mechanics of individual cells with multiplexed RT-qPCR gene expression analysis on the same single cells. Surprisingly, the genes that most strongly correlated with mechanical properties were not cytoskeletal, but rather were markers of extracellular matrix remodeling, epithelial-to-mesenchymal transition, cell adhesion, and cancer stemness. In addition, dimensionality reduction analysis showed that cell clustering was improved by combining mechanical and gene expression data types. The single cell genomechanics method demonstrates how single cell studies can identify molecular drivers that could affect the biophysical processes underpinning metastasis.

Synergic Action of Insulin-like Growth Factor-2 and miRNA-483 in Pterygium Pathogenesis.

Pterygium is a multifactorial disease in which UV-B is speculated to play a key role by inducing oxidative stress and phototoxic DNA damage. In search for candidate molecules that are useful for justifying the intense epithelial proliferation observed in pterygium, our attention has been focused on Insulin-like Growth Factor 2 (IGF-2), mainly detected in embryonic and fetal somatic tissues, which regulate metabolic and mitogenic functions. The binding between IGF-2 and its receptor Insulin-like Growth Factor 1 Receptor (IGF-1R) activates the PI3K-AKT pathway, which leads to the regulation of cell growth, differentiation, and the expression of specific genes. Since IGF2 is regulated by parental imprinting, in different human tumors, the IGF2 Loss of Imprinting (LOI) results in IGF-2- and IGF2-derived intronic miR-483 overexpression. Based on these activities, the purpose of this study was to investigate the overexpression of IGF-2, IGF-1R, and miR-483. Using an immunohistochemical approach, we demonstrated an intense colocalized epithelial overexpression of IGF-2 and IGF-1R in most pterygium samples (Fisher's exact test, p = 0.021). RT-qPCR gene expression analysis confirmed IGF2 upregulation and demonstrated miR-483 expression in pterygium compared to normal conjunctiva (253.2-fold and 12.47-fold, respectively). Therefore, IGF-2/IGF-1R co-expression could suggest their interplay through the two different paracrine/autocrine IGF-2 routes for signaling transfer, which would activate the PI3K/AKT signaling pathway. In this scenario, miR-483 gene family transcription might synergically reinforce IGF-2 oncogenic function through its boosting pro-proliferative and antiapoptotic activity.

Direct lysis of 3D cell cultures for RT-qPCR gene expression quantification

In vitro cell culture experiments are widely used to study cellular behavior in most biological research fields. Except for suspension cells, most human cell types are cultured as adherent monolayers on a plastic surface. While technically convenient, monolayer cultures can suffer from limitations in terms of physiological relevance, as their resemblance to complex in vivo tissue structures is limited. To address these limitations, three-dimensional (3D) cell culture systems have gained increased interest as they mimic key structural and functional properties of their in vivo tissue counterparts. Nevertheless, protocols established on monolayer cell cultures may require adjustments if they are to be applied to 3D cell cultures. As gene expression quantification is an essential part of many in vitro experiments, we evaluated and optimized a direct cell lysis, reverse transcription and qPCR protocol applicable for 3D cell cultures. The newly developed protocol wherein gene expression is determined directly from crude cell lysates showed improved cell lysis compared to the standard protocol, accurate gene expression quantification, hereby avoiding time-consuming cell harvesting and RNA extraction.

Gene Expression Study of Host and Mycobacterium tuberculosis Interactions in the Manifestation of Acute Tuberculosis.

Mycobacterium tuberculosis (M.tb) could induce type IV hypersensitivity. The chemotaxis of the leukocytes toward the site of infection and producing matrix metalloproteinases (MMPs) are key factors in the immune pathogenesis of tuberculosis (TB). Mononuclear cells were isolated from bronchoalveolar lavage (BAL) specimens, and the target from genomic DNA was used for qPCR TB diagnosis and cDNA for specific RT-qPCR gene expression. The subjects were then classified into TB+ and TB- groups, and the expression levels of CFP-10, ESAT-6, CCR1, CCR12 and MMP3,9 were evaluated. The mean level of CCR1 expression in TB+ and TB- patients' BAL was 1.71 ± 0.78 and 0.5 ± 0.22, respectively, which was statistically different (p = 0.01). The CCR2 level, in TB+ (2.07 ± 1.4), was higher than in TB- patients (1.42 ± 0.89, p = 0.01). The MMP9 expression in TB+ was 2.56 ± 0.68, also higher than in TB- patients (1.13 ± 0.35), while MMP3 was lower in TB+ (0.22 ± 0.09) than in TB- (0.64 ± 0.230, p = 0.05). The CCR2/CCR1 and MMP3/MMP9 balance in TB+ were reduced, compared to the TB-. The CFP-10 and ESAT-6 were highly expressed in TB+ patients. The CFP-10 expression had a strong negative correlation with albumin (r = - 0.93, p = 0.001), and a negative correlation with neutrophil (r = - 0.444, p = 0.1 with 90% CI). The MMP-9 expression showed a positive correlation with WBC count (r = 0.61, p = 0.02), in TB+, and had a negative correlation with BMI (r = 0.59, p = 0.02) in TB-. The M.tb CFP-10 might be implicated in lowering CCR2 and MMP3 expression in favour of M.tb dissemination. Moreover, the balance of CCR2/CCR1 and MMP3/MMP9 can be used as prognostic factors in the severity of TB.

Anti-inflammatory and Cytokines Modulatory Activities of Spondias mombin Linn. (Anacardiacea) in Wound Healing: Roles of IL6

Health problems relating to wound healing remain a significant issue in health management. Various formulations of Spondias mombin Linn. (Anacardiaceous) serves potent roles in ethnomedicine. To explore the counter-inflammatory and cytokines modulatory roles together with the antioxidant and wound healing properties of Spondias mombin Linn. (Anacardiaceous) leaves essential oil (SMEO) on excised wounds. Spondias mombin Linn. (Anacardiaceous) leaves essential oil was isolated through hydro distillation essential in Clevenger type apparatus (Borosil, India). Then thirty-eight healthy adult male albino rats (250 ± 20g) were grouped randomly into (n=6); Group 1- 50 µL 1% SMEO, Group 2- 0.1% DMSO and Tween 20 (Control), Group 3- Dermazin® ointment, Group 4-untreated, Group 5-50µL 10% SMEO, Group 6- 50µL 15% SMEO, with two unwounded samples, were treated for 14 days. Tissues of two rats was harvested per group on the 3 rd, 10th and 14th days after excision, wounded areas were excised for RT-qPCR cytokines analysis and gene expression (Light Cycler, Mannheim, Germany) (IL 6, IL1β, TNFα). The SMEO of (25–100 μg/ml) was passed through Total antioxidant/flavonoid/phenol phytochemical estimation assays. The assays showed high presence of flavonoids/phenols. The 10% and 15% essential oil healing progression showed efficiency over the standard. Cytokine analysis/gene expression revealed a significant reduction in IL6 levels, a key regulator of other pro-inflammatory cytokines and reparative process, after the 3rd, 7th and 14th day. The extracted Spondias mombin Linn. essential oil showed stimulation of an anti-inflammatory effect through a significant reduction of IL-6 levels as compared to the vehicle and standard treatment on the 3 rd, 10th through to the 14th day of the treatment course, neo-vascularization, tissue regeneration phase and overall improved wound healing better than the standard.

Rat liver ECM incorporated into electrospun polycaprolactone scaffolds as a platform for hepatocyte culture.

Liver disease is expanding across the globe; however, health-care systems still lack approved pharmaceutical treatment strategies to mitigate potential liver failures. Organ transplantation is the only treatment for liver failure and with increasing cases of liver disease, transplant programs increasingly cannot provide timely transplant availability for all patients. The development of pharmaceutical mitigation strategies is clearly necessary and methods to improve drug development processes are considered vital for this purpose. Herein, we present a methodology for incorporating whole organ decellularised rat liver ECM (rLECM) into polycaprolactone (PCL) electrospun scaffolds with the aim of producing biologically relevant liver tissue models. rLECM PCL scaffolds have been produced with 5w/w% and 10w/w% rLECM:PCL and were analyzed by SEM imaging, tensile mechanical analyses and FTIR spectroscopy. The hepatocellular carcinoma cell line, HepG2, was cultured upon the scaffolds for 14 days and were analyzed through cell viability assay, DNA quantification, albumin quantification, immunohistochemistry, and RT-qPCR gene expression analysis. Results showed significant increases in proliferative activity of HepG2 on rLECM containing scaffolds alongside maintained key gene expression. This study confirms that rLECM can be utilized to modulate the bioactivity of electrospun PCL scaffolds and has the potential to produce electrospun scaffolds suitable for enhanced hepatocyte cultures and in-vitro liver tissue models.

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome.

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.

Validation of reference genes for the normalization of RT-qPCR gene expression in Bacillus siamensis 1021 grown in different culture media.

Background and Objectives:House-keeping genes are generally selected as reference genes in gene expression analysis. However, some genes may not be stably expressed across all experimental conditions. Thus, this study aimed to validate seven house-keeping genes for gene expression analysis in Bacillus siamensis 1021.Materials and Methods:Strain 1021 was grown in potato dextrose broth, nutrient broth and mineral salt medium. Reverse-transcription quantitative PCR was used to determine Cq values of seven reference genes including gyrA, gyrB, ssb and dnaB, rpsU, gat_Yqey and udp in these media. Expression stability of these genes was analyzed, using geNorm and Normfinder applications. The target gene ftsZ was used for assessment of the best candidate genes.Results:Based on geNorm and Normfinder, ssb was the most-stably expressed gene, while udp was the least-stably expressed gene. Pairwise variation indicated the combination of ssb, gyrA, gyrB and gatB_Yqey was suitable for the normalization of ftsZ expression. ftsZ expression in potato dextrose broth and mineral salt medium was higher than that in nutrient broth. In contrast, the normalization against udp resulted in an under- and overestimation of ftsZ expression in potato dextrose broth and mineral salt medium, respectively.Conclusion:The combination of ssb, gyrA, gyrB and gatB_Yqey was the best candidate for normalization of target gene expression in B. siamensis 1021 in these media. This study emphasized the significance of reference gene validation for gene expression analysis and provided a guideline for future gene expression studies in B. siamensis.

A Two-Part Mixed Model for Differential Expression Analysis in Single-Cell High-Throughput Gene Expression Data.

The high-throughput gene expression data generated from recent single-cell RNA sequencing (scRNA-seq) and parallel single-cell reverse transcription quantitative real-time PCR (scRT-qPCR) technologies enable biologists to study the function of transcriptome at the level of individual cells. Compared with bulk RNA-seq and RT-qPCR gene expression data, single-cell data show notable distinct features, including excessive zero expression values, high variability, and clustered design. We propose to model single-cell high-throughput gene expression data using a two-part mixed model, which not only adequately accounts for the aforementioned features of single-cell expression data but also provides the flexibility of adjusting for covariates. An efficient computational algorithm, automatic differentiation, is used for estimating the model parameters. Compared with existing methods, our approach shows improved power for detecting differential expressed genes in single-cell high-throughput gene expression data.