Suitable Reference Research Articles

Quantifying tau deposition with the Parametric Estimation of Reference Signal Intensity: a 18F‐PI2620 tau‐PET and 4R‐tauopathy study

AbstractBackgroundTau positron emission tomography (PET) can detect abnormal tau in neurodegenerative diseases. Conventional reference regions show high PET signal variability and are susceptible to contamination from partial‐volume effects of tracer uptake. The parametric estimation of reference signal intensity (PERSI) has been introduced as a new method to overcome those issues in patients with Alzheimer’s disease. The current study investigates its applicability on patients with underlying 4R‐tauopathy with the radiotracer 18F‐PI‐2620.MethodIn this preliminary, cross‐sectional analysis, 9 patients with clinically suspected 4R‐tauopathy underwent T1‐weighted MRI and 0‐60 min dynamic 18F‐PI‐2620 PET scanning. PERSI identified voxels within the white matter without specific binding for use as a reference region, which was compared with conventional anatomically defined reference regions. Time‐activity curves (TAC) and standardized uptake value ratios in the time window of 20‐40 min (SUVr20‐40) were generated for regions of interest (frontal lobe and globus pallidum). Data was analysed with FSL, PETSurfer, and R.ResultSUVr20‐40 in the globus pallidum were generally higher than in the frontal lobe for all reference regions (p < 0.001). Frontal lobe, but not globus pallidum SUVr20‐40 were lower when derived from PERSI than when using the cerebellum (p < 0.001) or pons (p < 0.001). When comparing TACs, the PERSI ROI did not show the rapid peak (perfusion) as seen in all other regions (both target and reference), resulting in an overestimation of SUVR in the early phases of the scan. However, activity was the same for all reference regions at later timepoints resulting in similar SUVr20‐40 measures in the globus pallidum.ConclusionPERSI derived reference regions do not provide an accurate estimate of early phase perfusion and as such are not appropriate for use as reference regions for reference tissue modelling of PET data. Nonetheless, PERSI provides a suitable reference region for late phase SUVR calculations which is particularly relevant for diseases and tracers were an anatomically defined reference region cannot be easily determined. Future analyses on a larger cohort will determine effect sizes and ROCs to see whether PERSI is a suitable semi‐quantitative investigation to compare between different methodologies and diagnostic groups.

A High-Resolution Site Amplification Map for Wellington, New Zealand

Abstract Although earthquake site effects play a crucial role in the evaluation of local seismic hazard and associated risk, their quantification over the frequency range of interest for engineering applications still remains challenging. Mapping the local amplification at high resolution is difficult even in seismically active cities such as Wellington, New Zealand. Employing traditional methods to map amplification, such as the standard spectral ratio (SSR), is realistic only with sufficient density of strong-motion stations (SMS) across the city and the presence of a suitable rock reference station. Recently, hybrid standard spectral ratio methodologies (SSRh) have been proposed to fill in the gaps and provide estimates at much finer spatial resolution. SSRh combines traditional SSR, calculated on earthquake data between a soil reference and a rock station, with SSR computed from simultaneous ambient vibration recordings (SSRn) at a temporary location and the soil reference site within the sedimentary basin. In the last decade, over 450 single-station ambient noise measurements were undertaken across Wellington, and no collocated soil reference station is available, making the SSRh method as it stands impossible to apply. To overcome this limitation, we propose an adaptation of SSRh to capture the same basin response between a soil site and soil reference station as in the case of the synchronous ambient vibration data. We employ an additional interim step that uses the traditional SSRn between each of the soil sites and a rock reference broadband station recording synchronous long-term ambient vibration. The resulting empirical amplification model using the SSRh adaptation is in good agreement with the available SSR at SMS. Amplification factors up to 10 are present along the Centreport area, where significant damage was observed during the Mw 7.8 Kaikōura earthquake. By employing the adjusted SSRh methodology, we were able to develop a first-level high-resolution empirical site amplification model for Wellington. The approach provides an attractive solution for the evaluation of site effects across regions where a significant number of unsynchronized ambient vibration measurements are available.

Screening and Identifying Reference Genes for Erythrocyte Production from Cord Blood CD34+ Cells Exposed to Hypoxia.

Cord blood (CB) CD34+ cells have the potential to be used to achieve artificial hematopoiesis because of their ability to expand and differentiate in multiple directions. However, the mechanism and molecular changes underlying such differentiation are still unclear. The differentiation of CB CD34+ cells is generally driven by subtle changes in gene expression. A crucial method for examining gene expression is quantitative real-time polymerase chain reaction, but the accuracy of the results is dependent on the use of reliable reference genes. Here, the transcription levels of 10 novel candidate reference genes (EIF4G2, DYNC1H1, LUC7L3, CD46, POLR1D, WSB1, GAPVD1, HGS, LGALS8, and RBM5) and 8 traditional reference genes (GAPDH, YWHAZ, ACTB, B2MG, TBP, HMBS, PPIA, HPRT1) in CB CD34+ cells under different oxygen concentrations were screened and evaluated by using the geNorm and NormFinder algorithms. Comprehensive analysis conducted by RefFinder online tool showed that TBP (a traditional reference gene) and EIF4G2 (a novel reference gene) had the most stable expression, whereas GAPDH and HMBS were the least suitable reference genes under these conditions. These results may serve as a basis for selecting reference genes with stable expression for more accurate normalization under different oxygen concentration stimulation during CB CD34+ cells differentiation.

New Potential Sulfide Reference Materials for Microbeam S‐Fe‐Cu Isotope Measurements

S‐Fe‐Cu isotope systems are powerful tracers for revealing geochemical processes. However, the microanalysis of S‐Fe‐Cu isotopes is critically limited by the lack of suitable reference materials. Herein, we present three potential reference materials Ll‐Cpy (chalcopyrite), Ll‐Po (pyrrhotite) and Ll‐Sp (sphalerite) for in situ S‐Fe‐Cu isotope measurements. Numerous in situ S‐Fe‐Cu isotope measurements were performed over two years to assess isotopic homogeneity. The bulk S isotopic compositions were determined independently in seven laboratories by isotope ratio mass spectrometry (IRMS); the preferred δ34SV‐CDT for Ll‐Cpy, Ll‐Po, Ll‐Sp are 6.13 ± 0.37‰ (2s), 6.42 ± 0.37‰ (2s) and 6.28 ± 0.38‰ (2s), respectively. The bulk Fe isotope ratios in Fe‐bearing Ll‐Cpy and Ll‐Po were determined using solution nebulisation multi‐collector inductively coupled plasma‐mass spectrometry, and the obtained δ56FeIRMM‐014 values are 0.57 ± 0.07‰ (2s) and ‐0.62 ± 0.07‰ (2s), respectively. The mean bulk δ65CuNIST SRM 976 value of Ll‐Cpy is 0.57 ± 0.06‰ (2s). All the bulk values are in good agreement with the long‐term statistical results of laser ablation‐MC‐ICP‐MS and proposed as the recommended values. These sulfides are well characterised and isotopically homogeneous (at 30–40 μm spatial resolution), and can be used as potential calibration materials for in situ S‐Fe‐Cu isotope measurements.

Noise Source Diagnosis Method Based on Transfer Path Analysis and Neural Network

Clarifying the noise source and the contribution of each path is essential for the system’s noise control. The auxiliary converter cabinet, which is a crucial component of rail transportation, has numerous intricate noise sources. The contribution of each path point must be inverted-solved using known transfer functions and target point test values when identifying noise sources. This article suggests a method for diagnosing noise using transfer path analysis and neural networks (TPA-NN). Firstly, the principle and scheme for analyzing the transmission path of the converter cabinet are proposed. The transfer function of each path is obtained by selecting suitable path points, reference points, and target points for air and structure acoustic vibration experiments. The external target point data are then combined with the neural network’s linear fitting function, and the contribution of each path is used as an output for network training while some path point contributions are rebuilt. The results indicate that the method’s outcomes are most accurate when the converter cabinet’s path point is 13 and the target point is 6. This approach offers an innovative technique for locating noise sources in intricate systems.

Suitability of 210Pbex, 137Cs and 239+240Pu as soil erosion tracers in western Kenya

Land degradation resulting from soil erosion is a global concern, with the greatest risk in developing countries where food and land resources can be limited. The use of fallout radionuclides (FRNs) is a proven method for determining short and medium-term rates of soil erosion, to help improve our understanding of soil erosion processes. There has been limited use of these methods in tropical Africa due to the analytical challenges associated with 137Cs, where inventories are an order of magnitude lower than in the Europe. This research aimed to demonstrate the usability of 239+240Pu as a soil erosion tracer in western Kenya compared to conventional isotopes 210Pbex and 137Cs through the determination of FRN depth profiles at reference sites. Across six reference sites 239+240Pu showed the greatest potential, with the lowest coefficient of variation and the greatest peak-to-detection limit ratio of 640 compared to 5 and 1 for 210Pbex and 137Cs respectively. Additionally, 239+240Pu was the only radionuclide to meet the ‘allowable error’ threshold, demonstrating applicability to large scale studies in Western Kenya where the selection of suitable reference sites presents a significant challenge. The depth profile of 239+240Pu followed a polynomial function, with the maximum areal activities found between depths 3 and 12 cm, where thereafter areal activities decreased exponentially. As a result, 239+240Pu is presented as a robust tracer to evaluate soil erosion patterns and amounts in western Kenya, providing a powerful tool to inform and validate mitigation strategies with improved understanding of land degradation.

Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses

Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean–Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.

Suitable reference gene for silencing methods using microRNA encapsulated nanoparticles chitosan for the ovarian cancer cell line

MicroRNAs (miRNAs) are key regulators of gene expression, and their accurate quantification is essential for elucidating miRNA functional roles. Quantitative reverse transcription PCR (qRT-PCR) is widely utilized for miRNA expression analyses, although appropriate data normalization is critical for generating reliable results. The present study aimed to identify a suitable reference gene for normalizing miRNA qRT-PCR data in the ovarian cancer cell line SKOV-3. SKOV-3 cells were treated with miRNA-loaded chitosan nanoparticles or left untreated as a control. Total RNA, including small RNAs, was extracted from SKOV-3 cells and assessed for quality. The expression stability of 15 candidate reference genes was evaluated by GeNorm analysis. miR-584-5p was identified as the most stably expressed reference gene. To validate miRNA profiling, miR-584-5p was used to normalize expression levels, revealing distinct patterns of differential miRNA expression. Our findings provide critical insights into aberrant miRNA expression in ovarian cancer cells and underscore proper normalization as essential for accurate quantification of miRNAs by qRT-PCR.

52 The Power of Pangenome Assemblies in Ruminants

Abstract High quality reference genomes are the bases of all genomic research and they have facilitated a wealth of discoveries and research tools for the improvement of livestock species. These works have enabled major advances in the fields of animal science and genetics and impacted livestock production and associated industries for the benefit of society. However, because a single reference genome fails to represent all of the genetic material present across populations, it inherently limits the ability to comprehensively evaluate all genomic variants within a species. A complete understanding of how small and large genomic variants, from single nucleotide polymorphisms (SNP) to larger structural variations (SV) including insertions/deletions (InDels) and copy number variation (CNVs), have been selected for over time for specific environments and phenotypes, remains elusive. To address this, it is necessary to generate a catalog of genomic variants in populations subjected to natural or artificial selection pressures and evaluate their relationship among phenotypic classes. The pangenome approach is a means to comprehensively identify and catalog genomic variation and representation that will make more suitable reference for mapping population sequence data for variant detection and frequency evaluation. The goal of the most pangenome projects is to provide genomic resources to represent the immense genetic diversity of this species more completely. This will increase our understanding of genetic variation and its role in dictating different phenomes, empowering scientists to predict the physiological outcomes more effectively from genomic variation towards the improvement of breeding strategies. These pangenomes can then be used to discover the underlying mechanism of biological traits and inform future breeding strategies and support sustainable production of this global source of food and fiber.

Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress.

Chenopodium quinoa Willd. (quinoa), a member of the Amaranthaceae family, is an allotetraploid annual plant, endemic to South America. The plant of C. quinoa presents significant ecological plasticity with exceptional adaptability to several environmental stresses, including salinity. The resilience of quinoa to several abiotic stresses, as well as its nutritional attributes, have led to significant shifts in quinoa cultivation worldwide over the past century. This work first defines germination sensu stricto in quinoa where the breakage of the pericarp and the testa is followed by endosperm rupture (ER). Transcriptomic changes in early seed germination stages lead to unstable expression levels in commonly used reference genes that are typically stable in vegetative tissues. Noteworthy, no suitable reference genes have been previously identified specifically for quinoa seed germination under salt stress conditions. This work aims to identify these genes as a prerequisite step for normalizing qPCR data. To this end, germinating seeds from UDEC2 and UDEC4 accessions, with different tolerance to salt, have been analyzed under conditions of absence (0 mM NaCl) and in the presence (250 mM NaCl) of sodium chloride. Based on the relevant literature, six candidate reference genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Monensin sensitivity1 (MON1), Polypyrimidine tract-binding protein (PTB), Actin-7 (ACT7), Ubiquitin-conjugating enzyme (UBC), and 18S ribosomal RNA (18S), were selected and assessed for stability using the RefFinder Tool encompassing the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt in the evaluation. The data presented support the suitability of CqACT7 and CqUBC as reference genes for normalizing gene expression during seed germination under salinity stress. These recommended reference genes can be valuable tools for consistent qPCR studies on quinoa seeds.

Selection and Validation of Reference Genes for qRT-PCR Analysis of Gene Expression in Tropaeolum majus (Nasturtium)

Tropaeolum majus (nasturtium) is an important ornamental and medicinal plant due to its colorful flowers, shield-shaped leaves, and richness in mineral elements and bioactive compounds. However, the key genes related to these important biological traits, as well as their expression patterns and functions, remain obscure. In this study, to choose appropriate reference genes for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, we screened 14 candidate genes from the transcriptome of T. majus and evaluated their expression stability. Through evaluation with four commonly used algorithms (geNorm, NormFinder, BestKeeper, and RefFinder), EXP1, EXP2, and TUB6 were found to be the most stably expressed genes among different organs, while EXP1 combined with CYP2 was identified as the optimal reference gene combination for seeds at different development stages. For all the tested samples, EXP1, EXP2, CYP2, and ACT2 were the most suitable reference genes. Moreover, the target gene KCS11 involved in very-long-chain fatty acid biosynthesis was employed to confirm the most and least stable reference genes in different organs, seeds at different development stages, and all the tested samples. The expression profiles of KCS11 were similar, with minor differences based on the analysis of different stable reference genes (either alone or in combination), while the expression profiles were diverse and the relative expression level was overestimated when using the least stable ones. These results suggest that the appropriate selection of reference genes is critical for the normalization of gene expression. Furthermore, the reference genes screened in this study will greatly improve the accuracy of the qRT-PCR quantification of candidate genes involved in the many biological characteristics of nasturtium.

DNA Fingerprinting: A Milestone in Forensic Science

The successful interpretation of DNA in forensic science is contingent upon the significance of the evidence found at a crime scene and the availability of suitable reference samples.

Selection and validation of optimal reference genes for RT-qPCR analyses in Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae).

Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions (developmental stages, adult tissues, sugar treatment, and starvation treatment) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, β-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza.

Geochemical fingerprinting to determine sediment source contribution and improve contamination assessment in mining-impacted floodplains in the Philippines

Constraining the main sediment sources and pathways across landscapes impacted by anthropogenic activity is essential to limit the dispersal of sediment-borne contaminants, especially in global conservation priority areas. This study examined the provenance, partitioning, and enrichment of metals in the floodplain of the mining-affected Santa Cruz catchment, The Philippines. Composite geochemical fingerprinting of fine sediment samples (n = 36) was performed using a stepwise statistical screening procedure (range test, Kruskal-Wallis H test, discriminant function analysis) to derive the optimum set of tracers. Using a standard unmixing model, flood deposits downstream of the mining areas were shown to be predominantly mining-induced (71.9 ± 7.7 %), followed by natural erosion from gullies and stream banks (15.1 ± 11.0 %) and agricultural sediment (13.0 ± 5.1 %). Element partitioning data (Log Kd = 1.3–6.6) during a high flow event indicated that metals are dominantly associated and transported via suspended particulate matter. Background concentrations of Ni and Cr were found to be orders of magnitude higher than the threshold values set by international sediment quality guidelines (SQGs), emphasizing the need for site-specific SQGs in mineralised areas. Enrichment factor values indicated low to significant contamination of flood deposits relative to natural and agricultural sediment (EF 1.0–5.5). Furthermore, it was demonstrated that using different conservative elements could considerably influence the enrichment factor calculation. Based on tracer screening tests, Th was shown to be the most suitable reference element. The findings provide new insights on the application of geochemical tracers in a mining setting and integrating fingerprinting approaches with traditional assessment techniques to improve the reliability of contamination risk assessment in other areas facing similar sediment pollution problems.

Selection and Validation of Reference Genes for Reverse-Transcription Quantitative PCR Analysis in Sclerotium rolfsii.

Reference genes are important for the accuracy of gene expression profiles using reverse-transcription quantitative PCR (RT-qPCR). However, there are no available reference genes reported for Sclerotium rolfsii; it actually has a pretty diverse and wide host range. In this study, seven candidate reference genes (UBC, β-TUB, 28S, 18S, PGK, EF1α and GAPDH) were validated for their expression stability in S. rolfsii under conditions of different developmental stages, populations, fungicide treatments, photoperiods and pHs. Four algorithm programs (geNorm, Normfinder, Bestkeeper and ΔCt) were used to evaluate the gene expression stability, and RefFinder was used to integrate the ranking results of four programs. Two reference genes were recommended by RefFinder for RT-qPCR normalization in S. rolfsii. The suitable reference genes were GAPDH and UBC across developmental stages, PGK and UBC across populations, GAPDH and PGK across fungicide treatments, EF1α and PGK across photoperiods, β-TUB and EF1α across pHs and PGK and GAPDH across all samples. Four target genes (atrB, PacC, WC1 and CAT) were selected for the validation of the suitability of selected reference genes. However, using one or two reference genes in combination to normalize the expression of target genes showed no significant difference in S. rolfsii. In short, this study provided reliable reference genes for studying the expression and function of genes in S. rolfsii.

A new fuzzy decoupled sliding mode control of flexible joint robotic manipulators based on the finite‐time observer in the presence of chaos with experimental validation

AbstractThis paper presents a new fuzzy non‐singular fast terminal decoupled sliding mode control based on the finite‐time extended state observer for trajectory tracking control of flexible joint robot manipulators under chaos and uncertainty. First, a suitable chaotic reference is used in a synchronization mechanism to create chaos in the dynamics. Then, the essential modifications made to the conventional decoupled sliding mode control are introduced. A new coupling variable and a non‐singular fast terminal sliding surface are proposed in combination with an extended state observer. A complete convergence analysis is also performed for all the sliding surfaces. Furthermore, two fuzzy systems are designed in such a way that not only they can eliminate the chattering phenomenon but can also improve the convergence time without increasing the control input in a voltage‐based manner. The mathematical proof guarantees the finite‐time asymptotic stability of the closed‐loop control system under the proposed scheme for a class of under‐actuated systems in the presence of structured and un‐structured uncertainties. Finally, some simulations and experimental implementations as hardware‐in‐the‐loop are performed to evaluate the proposed method's performance. The proposed controller is an effective method, as confirmed by the numerical simulation results and experimental validation.

The Tuscany Regional Network for rare diseases: from European Reference Networks’ experience to registry based organisation and management model for rare diseases

BackgroundIn the European Union, a disease is defined as rare when it affects fewer than 1 in 2000 people. Currently, there are up to 8000 described rare diseases (RDs), collectively affecting 30 million people in the European Union. In 2004 Tuscany region (Italy) established a Regional Network of hospital units to ensure highly specialised medical care in the field of RDs. Shortly after the Rare Diseases Registry of Tuscany (Registro Toscano Malattie Rare—RTMR) was implemented. Here we describe the analysis performed on RTMR data which has recently allowed to remap the Network based on European Reference Networks’ model.ResultsData analysis was performed on 60,367 cases registered in RTMR, regarding 628 RDs. Two-hundred and fifteen active presidia have been evaluated. The assignment of each RD to the suitable European Reference Network has been made considering not only the number of registered cases, certifications and treatment plans for each Regional Presidium but also the competence in multidisciplinary management of the patient, from diagnosis to treatment. This evaluation has led to the establishment of twenty-one Regional Coordination Centres. They aggregate and coordinate Hospital Units which diagnose and treat one or a group of related RDs. In case of wide groups of RDs, Clinical Subnets are instituted. Updated statistics regarding RDs in Tuscany, list of RDs and Coordination Centres, as well as information about single Presidia are published and freely available on a designated webpage. Regional Decrees are regularly updated according to the network evolution.ConclusionsThe Rare Diseases Regional Network in Tuscany, based on the ERN model, has played a pivotal role in enhancing RD management and research. The remapping has led to a dynamic system, following not only scientific research but also the development of Presidia’s expertise. By pooling resources and expertise, the network has improved the availability and accessibility of specialized care for patients with RDs. Collaborative efforts, data sharing, and standardized registries are crucial for advancing RD research, improving diagnosis and treatment, and ultimately enhancing the quality of life for individuals living with RDs.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken gastrointestinal tract

The application of reverse transcription quantitative real-time PCR technology for the production of gene tissue expression profiles is a widely employed approach in molecular biology research. It is imperative to ascertain internal reference genes that exhibit stable expression across diverse tissues to ensure the precision of tissue gene expression profiles. While there have been studies documenting the most suitable reference genes for various tissues in chickens, there is a dearth of research on the identification of reference genes in the gastrointestinal (GI) tract of chickens. This study utilized 4 different algorithms (Delta CT, BestKeeper, NormFinder, and Genorm) to assess the stability of 19 internal reference genes in various GI tract tissues, including individual GI tract tissues, the anterior and posterior GI tract, and the entire GI tissue. The RefFinder software was employed to comprehensively rank these genes. The research findings successfully identified the most appropriate internal reference genes for each type of GI tissue. Furthermore, TBP, DNAJC24, Polr2b, RPL13, andAp2m exhibited stable expression in the entire and posterior GI tract, whereas HMBS, TBP, Ap2m, GUSB, DNAJC24, and RPL13 demonstrated stable expression in the anterior GI tract. However, the internal reference genes commonly utilized, namely β-Actin, 18s RNA, and ALB, exhibit poor stability and are not advised for future investigations concerning gene expression in the GI region. Consequently, MUC2 and CDX1, 2 genes that specifically express in the gut, were chosen for examination to ascertain the stability of the aforementioned internal reference genes in this particular study. In summary, this study presents a relatively stable set of internal reference genes that can be employed to enhance the precision of quantifying mRNA expression levels in functional genes within the chicken GI tract.

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System.

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a "zero set" of potential reference gene candidates.

Precise identification of cell states altered in disease using healthy single-cell references

Joint analysis of single-cell genomics data from diseased tissues and a healthy reference can reveal altered cell states. We investigate whether integrated collections of data from healthy individuals (cell atlases) are suitable references for disease-state identification and whether matched control samples are needed to minimize false discoveries. We demonstrate that using a reference atlas for latent space learning followed by differential analysis against matched controls leads to improved identification of disease-associated cells, especially with multiple perturbed cell types. Additionally, when an atlas is available, reducing control sample numbers does not increase false discovery rates. Jointly analyzing data from a COVID-19 cohort and a blood cell atlas, we improve detection of infection-related cell states linked to distinct clinical severities. Similarly, we studied disease states in pulmonary fibrosis using a healthy lung atlas, characterizing two distinct aberrant basal states. Our analysis provides guidelines for designing disease cohort studies and optimizing cell atlas use.