Expressed Reference Genes Research Articles

Multiomic integration analysis identifies atherogenic metabolites mediating between novel immune genes and cardiovascular risk

BackgroundUnderstanding genetic-metabolite associations has translational implications for informing cardiovascular risk assessment. Interrogating functional genetic variants enhances our understanding of disease pathogenesis and the development and optimization of targeted interventions.MethodsIn this study, a total of 187 plasma metabolite levels were profiled in 4974 individuals of European ancestry of the GCAT| Genomes for Life cohort. Results of genetic analyses were meta-analysed with additional datasets, resulting in up to approximately 40,000 European individuals. Results of meta-analyses were integrated with reference gene expression panels from 58 tissues and cell types to identify predicted gene expression associated with metabolite levels. This approach was also performed for cardiovascular outcomes in three independent large European studies (N = 700,000) to identify predicted gene expression additionally associated with cardiovascular risk. Finally, genetically informed mediation analysis was performed to infer causal mediation in the relationship between gene expression, metabolite levels and cardiovascular risk.ResultsA total of 44 genetic loci were associated with 124 metabolites. Lead genetic variants included 11 non-synonymous variants. Predicted expression of 53 fine-mapped genes was associated with 108 metabolite levels; while predicted expression of 6 of these genes was also associated with cardiovascular outcomes, highlighting a new role for regulatory gene HCG27. Additionally, we found that atherogenic metabolite levels mediate the associations between gene expression and cardiovascular risk. Some of these genes showed stronger associations in immune tissues, providing further evidence of the role of immune cells in increasing cardiovascular risk.ConclusionsThese findings propose new gene targets that could be potential candidates for drug development aimed at lowering the risk of cardiovascular events through the modulation of blood atherogenic metabolite levels.

Identification of Reliable Reference Genes for Use in Gene Expression Studies in Rat Febrile Seizure Model.

The study of the pathogenesis of febrile seizures and their consequences frequently necessitates gene expression analysis. The primary methodology employed for such analysis is reverse transcription with quantitative polymerase chain reaction (RT-qPCR). To ensure the accuracy of data obtained by RT-qPCR, it is crucial to utilize stably expressed reference genes. The objective of this study was to identify the most suitable reference genes for use in the analysis of mRNA production in various brain regions of rats following prolonged neonatal febrile seizures. The expression stability of eight housekeeping genes was evaluated using the online tool RefFinder in the dorsal and ventral hippocampal regions and in the temporal and medial prefrontal cortex of the brain. The Ppia gene exhibited the greatest stability of expression. Conversely, the genes with the least stable expression levels were Actb and Ywhaz; thus, it is not recommended to use them for normalization in a febrile seizure model. Additionally, the majority of housekeeping genes demonstrate age-related, region-specific fluctuations. Therefore, it is crucial to employ the appropriate housekeeping genes for each brain structure under investigation when examining the expression dynamics of genes of interest in a febrile seizure model.

Stability assessment of housekeeping genes for qRT-PCR in Yersinia enterocolitica cultured at 22°C and 37°C.

Yersinia enterocolitica, a species within the genus Yersinia, thrives optimally at 22-25°C but can also grow at the mammalian core body temperature of 37°C. This dual temperature adaptability necessitates establishing both temperature conditions in research to examine the effects on various biological processes. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the lack of alternatives and information often leads to the default use of the 16S rRNA gene despite potential limitations. This investigation sourced 16 potential reference genes through a comprehensive review of the literature and transcriptome sequencing data analysis. We validated the expression stability of these genes via qRT-PCR across 12 Y. enterocolitica strains, representing the four prevalent serotypes O:3, O:5,27, O:8, and O:9, isolated from diarrheal patient stool samples. This approach aimed to minimize the impact of serotype heterogeneity. After acquiring Cq values, gene stability was evaluated using four established algorithms-ΔCq, geNorm, NormFinder, and BestKeeper-and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method. Our study suggests that the genes glnS, nuoB, glmS, gyrB, dnaK, and thrS maintain consistent expression across varying culture temperatures, supporting their candidacy as robust housekeeping genes. We advise against the exclusive use of 16S rRNA for this purpose. Should tradition prevail in its utilization, it must be employed with discernment, preferably alongside one or two of the housekeeping genes identified in this study as internal controls.IMPORTANCEIn our study, we focused on identifying stable reference genes for quantitative real-time PCR (qRT-PCR) experiments on Y. enterocolitica cultured at different temperatures (22°C and 37°C). After thoroughly evaluating 16 candidate genes, we identified six genes-glnS, nuoB, glmS, gyrB, dnaK, and thrS-as exhibiting stable expression across these temperature conditions, making them ideal reference genes for Y. enterocolitica studies. This discovery is crucial for ensuring the accuracy and reliability of qRT-PCR data, as the choice of appropriate reference genes is key to normalizing expression data and minimizing experimental variability. Importantly, our research extended beyond bioinformatics analysis by incorporating validation with clinical strains, bridging the gap between theoretical predictions and practical application. This approach not only underscores the robustness and reliability of our findings but also directly addresses the critical need for experimental validation in the field. By providing a set of validated, stably expressed reference genes, our work offers valuable guidance for designing experiments involving Y. enterocolitica, enhancing the reliability of research outcomes, and advancing our understanding of this significant pathogen.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken liver tissues and LMH cells

The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.

Birc5 and Nudc are screened as candidate reference genes for RT-qPCR studies in mouse cementoblast mineralization using time-series RNA-seq data.

The robustness and credibility of RT-qPCR results are critically dependent on the selection of suitable reference genes. However, the mineralization of the extracellular matrix can alter the intracellular tension and energy metabolism within cells, potentially impacting the expression of traditional reference genes, namely Actb and Gapdh. To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization. Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking. A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes. RefFinder's stability ranking does not consider the influence of primer efficiency. We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.

Reference Gene Selection and Gene Expression Analysis during Gall Development of Zizania latifolia

Reference Gene Selection and Gene Expression Analysis during Gall Development of Zizania latifolia

Assessment of reference genes for qRT-PCR normalization to elucidate host response to African swine fever infection.

Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

Time- and Region-Specific Selection of Reference Genes in the Rat Brain in the Lithium-Pilocarpine Model of Acquired Temporal Lobe Epilepsy.

Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) is a commonly used tool for gene expression analysis. The selection of stably expressed reference genes is required for accurate normalization. The aim of this study was to identify the optimal reference genes for RT-qPCR normalization in various brain regions of rats at different stages of the lithium-pilocarpine model of acquired epilepsy. We tested the expression stability of nine housekeeping genes commonly used as reference genes in brain research: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on four standard algorithms (geNorm, NormFinder, BestKeeper, and comparative delta-Ct), we found that after pilocarpine-induced status epilepticus, the stability of the tested reference genes varied significantly between brain regions and depended on time after epileptogenesis induction (3 and 7 days in the latent phase, and 2 months in the chronic phase of the model). Pgk1 and Ywhaz were the most stable, while Actb, Sdha, and B2m demonstrated the lowest stability in the analyzed brain areas. We revealed time- and region-specific changes in the mRNA expression of the housekeeping genes B2m, Actb, Sdha, Rpl13a, Gapdh, Hprt1, and Sdha. These changes were more pronounced in the hippocampal region during the latent phase of the model and are thought to be related to epileptogenesis. Thus, RT-qPCR analysis of mRNA expression in acquired epilepsy models requires careful selection of reference genes depending on the brain region and time of analysis. For the time course study of epileptogenesis in the rat lithium-pilocarpine model, we recommend the use of the Pgk1 and Ywhaz genes.

Selection of reference genes for normalizing gene expression data across seasons in spermatozoa of water buffalo (Bubalus bubalis).

Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls' spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated 'endoGene' pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that GAPDH and PP1A were the most stably expressed among the gene panel, whereas ATPSF1 and ACTB were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, HSFY2 and AKAP4, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.

Exploration of reference genes for the development of a diagnostic kit on vascular aging in human saliva.

Identifying reliable biomarkers in saliva can be a promising approach to developing a rapid diagnostic kit for detecting vascular aging. This study investigated the most suitable reference gene for polymerase chain reaction (PCR) in saliva that is not affected by vascular aging variables. Whole saliva samples were collected to assess the expression of reference genes: actin beta (ACTB), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The most abundantly expressed gene was 18S rRNA, and the least expressed gene was GAPDH. Four genes were ranked according to their relative stability, as determined by mathematical algorithms, indicating that ACTB and 18S rRNA were stably expressed as reference genes. 18S rRNA was identified as the most promising reference gene for detecting systemic diseases using saliva from patients with vascular aging in these limited experimental conditions.

Characterizing reference genes for high-fidelity gene expression analysis under different abiotic stresses and elicitor treatments in fenugreek leaves

BackgroundQuantifying gene expression is a critical aspect of applied genomics research across all organisms, and real-time PCR has emerged as a powerful tool for this purpose. However, selecting appropriate internal control genes for data normalization presents specific challenges. This study aimed to identify suitable reference genes for gene expression analysis under various conditions, encompassing salinity, low and high-temperature stresses, and different elicitor treatments. These treatments included titanium dioxide, cold plasma, 24-epibrassinolide, and melatonin, resulting in a total of 13 unique treatments and 148 treatment combinations applied to fenugreek plants.ResultsAs per the analysis performed with the BestKeeper tool, EEF-1α, and GAPDH were recognized as the most stable reference genes under the majority of conditions. Furthermore, the GeNorm and NormFinder tools identified β-tubulin and EEF-1α as the most stable reference genes. The findings of this research demonstrated that, although the stability of three reference genes expression was acceptable in almost all evaluated treatments, fluctuations in their expression were observed under the treatments of cold stress with TiO2 NPs application, cold plasma application with salinity stress, and cold plasma application with high-temperature stress compared to others. Simultaneously, the GeNorm analysis results demonstrated that in the mentioned treatments, relying on only one reference gene is inadequate. To corroborate the results, we examined the expression profile of the SSR gene, a pivotal gene in diosgenin biosynthesis, under all investigated treatments and treatment combinations. The outcomes suggested that employing stable reference genes yielded highly consistent results.ConclusionsThe varying expression patterns of the target genes emphasize the crucial need for precise optimization of experimental conditions and selecting stable reference genes to achieve accurate results in gene expression studies utilizing real-time PCR. These findings offer valuable insights into the selection of appropriate reference genes for gene expression analysis under diverse conditions using real-time PCR.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Assessment of Reference Genes Stability in Cortical Bone of Obese and Diabetic Mice.

Bone, a pivotal structural organ, is susceptible to disorders with profound health implications. The investigation of gene expression in bone tissue is imperative, particularly within the context of metabolic diseases such as obesity and diabetes that augment the susceptibility to bone fractures. The objective of this study is to identify a set of internal control genes for the analysis of gene expression. This study employs reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) to assess gene expression in bone tissue. We selected fourteen housekeeping genes and assessed their stability in the cortical bone of mouse models for obesity and diabetes using four well-established algorithms (GeNorm, BestKeeper, NormFinder, and the comparative Delta Ct method). We identified Rpl13a as the mostly stably expressed reference gene in cortical bone tissue from mouse models of obesity and diabetes (db/db), while Gapdh was found to be the most stable reference gene in another diabetes model, KKAy mice. Additionally, Ef1a, Ppia, Rplp0, and Rpl22 were identified as alternative genes suitable for normalizing gene expression in cortical bone from obesity and diabetes mouse models. These findings enhance RT-qPCR accuracy and reliability, offering a strategic guide to select reference gene for studying bone tissue gene expression in metabolic disorders.

Evaluation and normalization of a set of reliable reference genes for quantitative sgk-1 gene expression analysis in Caenorhabditis elegans-focused cancer research

Multiple signaling pathways have been discovered to play a role in aging and longevity, including the insulin/IGF-1 signaling system, AMPK pathway, TOR signaling, JNK pathway, and germline signaling. Mammalian serum and glucocorticoid-inducible kinase 1 (sgk-1), which has been associated with various disorders including hypertension, obesity, and tumor growth, limits survival in C. elegans by reducing DAF-16/FoxO activity while suppressing FoxO3 activity in human cell culture. C. elegans provides significant protection for a number of genes associated with human cancer. The best known of these are the lin-35/pRb (mammalian ortholog pRb) and CEP-1 (mammalian ortholog p53) genes. Therefore, in this study, we aimed to investigate the expression analyzes of sgk-1, which is overexpressed in many types of mammalian cancer, in mutant lin-35 and to demonstrate the validation of reference genes in wild-type N2 and mutant lin-35 for C. elegans-focused cancer research. To develop functional genomic studies in C. elegans, we evaluated the expression stability of five candidate reference genes (act-1, ama-1, cdc-42, pmp-3, iscu-1) by quantitative real-time PCR using five algorithms (geNorm, NormFinder, Delta Ct method, BestKeeper, RefFinder) in N2 and lin-35 worms. According to our findings, act-1 and cdc-42 were effective in accurately normalizing the levels of gene expression in N2 and lin-35. act-1 and cdc-42 also displayed the most consistent expression patterns, therefore they were utilized to standardize expression level of sgk-1. Furthermore, our results clearly showed that sgk-1 was upregulated in lin-35 worms compared to N2 worms. Our results highlight the importance of definitive validation using mostly expressed reference genes.

Gene expression profile of immune-check point in response to Trastuzumab therapy in patients with HER-2 positive breast cancer.

Aim: To clarify the association between response to Trastuzumab and molecular expression of TIM-3 and FOXP-3 immune checkpoints. Materials and Methods: FOXP-3 and TIM-3 expression in peripheral blood was analyzed using qPCR, and the serum level of Trastuzumab was estimated using an immune sorbent enzyme assay. Results: During treatment with Trastuzumab, the FOXP-3 gene expression showed a significant decline throughout one year of treatment, going from 0.85 at cycle 9 to 0.75 at cycle 17. While the TIM-3 gene expression showed a significant up regulation at cycle 9 to 2.8 fold, followed by a reduction in the fold change from 2.8 to 1.7 in the font of reference gene expression. Conclusions:FOXP-3 and TIM-3 have the potential to be suggestive markers that can anticipate the response to Trastuzumab, but they are not capable of predicting the likelihood of recurrence.

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.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

HArmonized single-cell RNA-seq Cell type Assisted Deconvolution (HASCAD)

BackgroundCell composition deconvolution (CCD) is a type of bioinformatic task to estimate the cell fractions from bulk gene expression profiles, such as RNA-seq. Many CCD models were developed to perform linear regression analysis using reference gene expression signatures of distinct cell types. Reference gene expression signatures could be generated from cell-specific gene expression profiles, such as scRNA-seq. However, the batch effects and dropout events frequently observed across scRNA-seq datasets have limited the performances of CCD methods.MethodsWe developed a deep neural network (DNN) model, HASCAD, to predict the cell fractions of up to 15 immune cell types. HASCAD was trained using the bulk RNA-seq simulated from three scRNA-seq datasets that have been normalized by using a Harmony-Symphony based strategy. Mean square error and Pearson correlation coefficient were used to compare the performance of HASCAD with those of other widely used CCD methods. Two types of datasets, including a set of simulated bulk RNA-seq, and three human PBMC RNA-seq datasets, were arranged to conduct the benchmarks.ResultsHASCAD is useful for the investigation of the impacts of immune cell heterogeneity on the therapeutic effects of immune checkpoint inhibitors, since the target cell types include the ones known to play a role in anti-tumor immunity, such as three subtypes of CD8 T cells and three subtypes of CD4 T cells. We found that the removal of batch effects in the reference scRNA-seq datasets could benefit the task of CCD. Our benchmarks showed that HASCAD is more suitable for analyzing bulk RNA-seq data, compared with the two widely used CCD methods, CIBERSORTx and quanTIseq. We applied HASCAD to analyze the liver cancer samples of TCGA-LIHC, and found that there were significant associations of the predicted abundance of Treg and effector CD8 T cell with patients’ overall survival.ConclusionHASCAD could predict the cell composition of the PBMC bulk RNA-seq and classify the cell type from pure bulk RNA-seq. The model of HASCAD is available at https://github.com/holiday01/HASCAD.

Insensitive Effects of Inflammatory Cytokines on the Reference Genes of Synovial Fluid Resident-Mesenchymal Stem Cells Derived from Rheumatoid Arthritis Patients.

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1β) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1β but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines.

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.