Foreign ownership, subjective willing, and internal control level.

Foreign shareholders are essential in the capital market. The study on A-share listed firms from 2012 to 2021 examines the impact of foreign ownership on internal control and its transmission effect. Using text analysis and machine learning methods, we construct a variable named "internal control willingness" to explore the impact of subjective willingness. The findings indicate that foreign shareholding significantly enhances internal control quality, with a more pronounced effect observed in samples demonstrating a more positive internal control willingness. Moreover, foreign shareholders contribute to the invested firm's sustainable development by enhancing internal control quality. Further research demonstrates that the positive impact of foreign shareholding is more significant in firms with legal foreign shareholders, highly competitive industries, and sound legal environments. These findings can aid host country firms in more efficiently leveraging foreign resources and provide empirical evidence for opening up China's capital market and formulating foreign investment regulations.

Does the disclosure of internal control material deficiency remediation information matter for ESG performance? Evidence from China.

We examine the effects of the disclosure of internal control material deficiency remediation (ICMDR) information on firms' environmental, social and governance (ESG) performance. Using a sample of Chinese listed firms that disclosed ICMD information in the period 2012-2021, we find that firms which disclose their remediation information show better ESG performance and this improvement is achieved by mitigating financial risk. The results hold for various endogeneity concerns. In addition, the signal transmission effects of ICMDR disclosure on ESG performance occur mainly in the social and governance dimensions, whereas the effect on the environmental dimension is insignificant. Further heterogeneity analysis shows that, remediation information disclosure strongly improves ESG performance in firms that actively disclose remediation information, as well as in state-owned and non-heavily polluting enterprises. This study adds to the existing literature by revealing the role of remediation information disclosure in ESG performance, which has been a less explored topic. It also analyses the mediation role of financial risk to inform enterprises to disclose their remediation information as a means of achieving better sustainable development.

Digital transformation and environmental, social, and governance greenwashing: Evidence from China.

This paper examines the relationship between digital transformation(DT) and environmental, social, and governance(ESG) greenwashing using Chinese listed companies as a sample from 2012 to 2022. Furthermore, it analyzes the enterprise and regional heterogeneity as well as the influencing mechanisms on this relationship. The research results indicate that corporate digital transformation significantly inhibits ESG greenwashing, with a more pronounced effect on companies in non-high-pollution industries, high-tech industries, and the eastern region. In addition, mechanism tests reveal that internal control and financing constraints play a partial mediating role. Digital transformation suppresses ESG greenwashing by enhancing the quality of internal control and alleviating financing constraints. The primary contribution of this paper lies in demonstrating that digital transformation can serve as a strategic tool to mitigate ESG greenwashing. This enriches the research on the outcomes of digital transformation as well as the factors influencing ESG greenwashing. The conclusions of this paper provide theoretical foundations and policy recommendations for better ESG development by enterprises and governments in emerging markets. At the same time, this paper has a certain guiding role for the introduction and implementation of policies to encourage digital transformation.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

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.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection.

RT-qPCR dissects transcription-based processes but requires reference genes (RGs) for data normalization. This study prospected RGs for mouse macrophages (pMØ) and spleen infected with Listeria monocytogenes. The pMØ were infected in vitro with L. monocytogenes or vehicle for 4 h. Mice were injected with L. monocytogenes (or vehicle) and euthanized 24 h post-injection. The RGs came from a multispecies primer set, from the literature or designed here. The RG ranking relied on GeNorm, NormFinder, BestKeeper, Delta-CT and RefFinder. B2m-H3f3a-Ppia were the most stable RGs for pMØ, albeit RG indexes fine-tuned estimations of cytokine relative expression. Actß-Ubc-Ppia were the best RGs for spleen but modestly impacted the cytokine relative expression. Hence, mouse models of L. monocytogenes require context-specific RGs for RT-qPCR, thus reinforcing its paramount contribution to accurate gene expression profiling.

Differences of circular RNA expression profiles between monozygotic twins' blood, with the forensic application in bloodstain and saliva.

Monozygotic twins (MZTs) possess identical genomic DNA sequences and are usually indistinguishable through routine forensic DNA typing methods, which can be relevant in criminal and paternity cases. Recently, novel epigenetic methods involving DNA methylation and microRNA analysis have been introduced to differentiate MZTs. In this study, we explore the potential of using epigenetic markers, specifically circular RNAs (circRNAs), a type of non-coding RNA (ncRNA), to identify MZTs, and investigate the unique expression patterns of circRNAs within pairs of MZTs, enabling effective differentiation. Epigenetics regulates gene expression at the post-transcriptional level and plays a crucial role in cell growth and aging. CircRNAs, a recently characterized subclass of ncRNA, have a distinct covalent loop structure without the typical 5' cap or 3' tail. They have been reported to modulate various cellular processes and play roles in embryogenesis and eukaryotic development. To achieve this, we conducted a comprehensive circRNA sequencing analysis (circRNA-seq) using total RNA extracted from the blood samples of five pairs of MZTs. We identified a total of 15,257 circRNAs in all MZTs using circRNA-seq. Among them, 3, 21, 338, and 2967 differentially expressed circRNAs (DEcircRNAs) were shared among five, four, three, and two pairs of MZTs, respectively. Subsequently, we validated twelve selected DEcircRNAs using real-time quantitative polymerase chain reaction (RT-qPCR) assays, which included hsa_circ_0004724, hsa_circ_0054196, hsa_circ_004964, hsa_circ_0000591, hsa_circ_0005077, hsa_circ_0054853, hsa_circ_0054716, hsa_circ_0002302, hsa_circ_0004482, hsa_circ_0001103, novel_circ_0030288 and novel_circ_0056831. Among them, hsa_circ_0005077 and hsa_circ_0004482 exhibited the best performance, showing differences in 7 out of 10 pairs of MZTs. These twelve differentially expressed circRNAs also demonstrated strong discriminative power when tested on saliva samples from 10 pairs of MZTs. Notably, hsa_circ_0004724 displayed differential expression in 8 out of 10 pairs of MZTs in their saliva. Additionally, we evaluated the detection sensitivity, longitudinal temporal stability, and suitability for aged bloodstains of these twelve DEcircRNAs in forensic scenarios. Our findings highlight the potential of circRNAs as molecular markers for distinguishing MZTs, emphasizing their suitability for forensic application.

Evaluation of the applicability of internal controls on self-collected samples for high-risk human papillomavirus is needed.

BACKGROUND: Self-collection of cervical samples to detect high-risk human papillomavirus (hr-HPV) is a trending topic in primary cervical cancer screening. This study evaluates the applicability of a self-sampling device to routine molecular procedures for hr-HPV detection. METHODS: In a primary health care facility in Kinshasa, Congo, 187 self-collected samples (Evalyn Brush) were gathered and sent to Ghent University Hospital (UZ Ghent) and Algemeen Medisch Labo (AML) in Belgium where routine tests for hr-HPV were applied (Abbott RealTime hr-HPV and qPCR (E6/E7), respectively). Sample type effect was evaluated by comparing the internal control (IC) between the self-collected samples and routine, clinician-taken samples randomly selected from the UZ Ghent archive. RESULTS: In UZ Ghent an error was encountered in 9.1% (17/187) of self-collected samples due to a lack of IC signal. The hr-HPV prevalence in the remaining 170 samples was 18,8%. Comparing IC results between the self-collected and clinician-collected groups, a significant difference (p < 0,001) was found, with higher IC signals in the clinician-collected group. In AML, an error was encountered in 17.6% (33/187) of samples, including 16/17 of the UZ Ghent. The remaining sample with IC error gave a negative result in AML. Among the 154 samples without IC error at AML, a correlation of 90% was seen between both laboratories with a 77% negativity rate. CONCLUSION: Testing the self-collected specimens by 2 routine hr-HPV tests gave a high IC error rate (9.1-17.6%). A possible solution would be to differentiate cut-offs for IC values depending on sample type, as currently used cut-offs are set for clinician-taken samples.

Optimizing internal control in public hospital supply chain: a game theory-based approach.

Objective: Our study aims to enhance the precision of internal control construction within public hospital supply chains and minimize the subjective bias influence. We have integrated the game theory combination weighting method into the design of internal control paths and based on this, developed a series of pioneering solutions. This innovative approach is anticipated to heighten the effectiveness and scientific rigor of the internal control design scheme within the supply chain. Method: Firstly, we utilized literature review and expert interviews to delve into the key factors of public hospital supply chain internal control, forming an index system for public hospital supply chain internal control that aligns with current informatization requirements. Subsequently, we incorporated the Game Theory Combination Weighting Method into this study. By means of the Analytic Hierarchy Process and the Entropy Weighting Method we determined the subjective and objective weights of each index and obtained their comprehensive weights through the Game Theory Combination Weighting Method. Then, based on the analysis results, we designed a series of internal control construction schemes and implemented these schemes at Weifang Maternal and Child Health Hospital between 2019 and 2023. Finally, using the Fuzzy Comprehensive Evaluation Method to assess and compare the actual effects before and after the implementation of the schemes, thereby validating the effectiveness of the Game Theory Combination Weighting Method in the design of the internal control path of public hospital supply chains. Results: The fuzzy comprehensive evaluation results for the years 2019 and 2023 demonstrated that after implementing our design schemes using the Game Theory Combination Weighting Method, the hospital's satisfaction in aspects such as plan-side control, purchase-side control, asset-side control, expenditure business control, and contract management control has significantly improved. Conclusion: Our research indicates that the Game Theory Combination Weighting Method is applicable to the path design of internal control links in public hospital supply chains. This method has effectively enhanced the targeted improvement of weak links within the construction of internal controls in the supply chain of public hospitals and is of great significance for improving the scientific nature of supply chain internal control management.

Pathogen quantitative efficacy of different spike-in internal controls and clinical application in central nervous system infection with metagenomic sequencing.

IMPORTANCE: Metagenomic next-generation sequencing (mNGS) has been used broadly for pathogens detection of infectious diseases. However, there is a lack of method for the absolute quantitation of pathogens by mNGS. We compared the quantitative efficiency of three mNGS internal controls (ICs) Thermus thermophilus, T1 phages, and artificial DNA sequence and developed the most applicable strategies for pathogen quantitation via mNGS in central nervous system infection. The IC application strategy we developed will enable mNGS analysis to assess the pathogen load simultaneously with the detection of pathogens, which should provide critical information for quick decision-making of treatment as well as clinical prognosis.

CRISPR/Cas12a-assisted visible fluorescence for pseudo dual nucleic acid detection based on an integrated chip.

BACKGROUND: A false negative result is one of the major problems in nucleic acid detection. Failure to screen positive samples for pathogens or viruses poses a risk to public health. This situation will lead to more serious consequences for infectious pathogens or viruses. At present, the common solution is to introduce exogenous or endogenous internal control. Because it amplifies and is detected separately from the target gene, it cannot avoid false negative results caused by DNA extraction failure or reagent inactivation. There is an urgent need for a simple and reliable method to solve the false negative problem of nucleic acid detection. RESULTS: We established a chip and an on-chip detection method for the integrated detection of target genes and internal control using the CRISPR system in LAMP amplification products. The chip is processed from a low-cost PMMA board and has three chambers and some channels. After adding the sample, the chip only needs to be rotated twice, and the sample enters three chambers successively depending on its gravity for dual LAMP reaction and CRISPR detections. With a portable LED blue light exciter, visual fluorescence detection is realized. Whether the detection result is positive, negative, or invalid can be determined according to the fluorescence in the CRISPR chamber for target gene and CRISPR chamber for internal control. In this study, the detection of Salmonella enterica in Fenneropenaeus chinensis was taken as an example. The results showed good specificity and sensitivity. It could detect as low as 15 copies/µL of Salmonella enterica. SIGNIFICANCE: The on-chip detection solves the problem of aerosol contamination and false negative results. It has the advantages of high sensitivity, high specificity, high accuracy, and low cost. This research will advance the development of nucleic acid detection technology, providing a new and reliable strategy for POCT detection of pathogenic bacteria and viruses.

Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand.

IMPORTANCE: Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, an environmental Gram-negative bacterium. Early detection of B. pseudomallei infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify B. pseudomallei in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect B. pseudomallei in all sample types, including samples from blood. We developed an optimal DNA extraction method for B. pseudomallei from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of B. pseudomallei infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of B. pseudomallei in clinical settings.

Does the implementation of green finance regulation promote the high-quality development of enterprises? Evidence from a quasi-natural experiment in China.

The improvement of enterprise total factor productivity and labor productivity is the micro-embodiment of high-quality economic development. Green finance relies on the dual functions of resource allocation and environmental regulation to guide enterprises to adjust their mode of operation through incentive and restraint mechanisms, attach importance to energy conservation and environmental protection, and guide enterprises to develop with high quality. Taking the construction of the green financial supervision system in 2016 as a quasi-natural experiment, we constructed a difference-in-difference model to investigate the impact and mechanism of green finance on the high-quality development of enterprises, based on the panel data of Chinese A-share listed companies from 2006 to 2020. The results show that the implementation of green finance effectively promotes the high-quality development of enterprises. This promotion effect is heterogeneous from perspectives of enterprise-specific characteristics, executive education background, and environmental regulation intensity. The influence mechanisms mainly rely on tightening financial constraints, upgrading the level of green technology innovation, and improving the quality of internal control. These findings provide an important decision-making reference for better implementing green finance policies and promoting high-quality economic development under the green and low-carbon concept and carbon peak carbon neutrality goals.

Internal controls driven by mindfulness toward enhanced ethical behaviors: Empirical evidence from Vietnam.

This study develops and tests a moderated mediation model regarding the effectiveness of internal control structure on organizational ethical behaviors via the mediating role of internal control effectiveness and the moderating role of organizational mindfulness in the relationship between internal control structure and internal control effectiveness. The proposed model and its hypotheses were tested using partial least squares structural equation modeling (PLS-SEM) in SmartPLS3 with survey data from 540 large Vietnamese manufacturing and service firms. This study found the following: (1) The internal control structure positively affects internal control effectiveness, which in turn promotes ethical organizational behaviors; and (2) The effect of internal control structure on internal control effectiveness is amplified by strengthening organizational mindfulness. The findings demonstrate that combining internal control systems and organizational mindfulness contributes to the ethical business practices of firms in an emerging market. Our study bridges the gap in the literature on internal control and mindfulness by providing empirical evidence on how the interaction between organizational mindfulness and internal control systems can promote ethical business practices. Additionally, our study advances the current understanding of how internal control systems can interact with organizational mindfulness to influence ethical business practices in the context of an emerging market.

COVID-19 PCR: frequency of internal control inhibition in clinical practice.

Introduction: Diagnosis of COVID-19 (coronavirus disease 2019) is best performed with real-time (quantitative) PCR (qPCR), the most sensitive method for detection and quantification of viral RNA. Using the Centers for Disease Control and Prevention (CDC) protocol, for each sample tested for the virus, three qPCR tests are performed, targeting the viral genes N1 and N2, in addition to the internal control gene RNase P. Samples in which internal control fails to amplify should be labelled 'invalid'. Methods: This study aims to determine the frequency of inhibition of the RNase P gene used as an internal control in qPCR tests for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in a reference hospital in Southern Brazil during the COVID-19 pandemic (1 February 2021 to 31 March 2021). Results: A total 10, 311 samples were available for analysis. The mean cycle threshold (Ct) value for the RNAse P gene was 26.65 and the standard deviation was 3.18. A total of 252 samples were inhibited (2.4%) during the study period: amongst these, 77 (30.5%) showed late amplifications (beyond 2 standard deviations from the mean Ct value), and 175 (69.4%) revealed no fluorescence at all for the RNase P gene. Conclusions: This study showed a low percentage of inhibition using RNase P as an internal control in COVID-19 PCRs using the CDC protocol, thus proving the effectiveness of this protocol for identification of SARS-CoV-2 in clinical samples. Re-extraction was efficacious for samples that showed little or no fluorescence for the RNase P gene.

Sistema automatizado para el control de medios de informática, ofimática y comunicaciones CIMIOC

Introducción: en las instituciones pertenecientes al Ministerio de Salud Pública de Cuba, la forma habitual de registrar el control de las Tecnologías de la Información y la Comunicación ha sido mediante hojas de Excel, procesadores de texto y fichas técnicas impresas. El control informatizado ofrece ventajas, pero las aplicaciones informáticas existentes a nivel mundial son costosas o no adaptables a los requerimientos. Objetivo: desarrollar una herramienta informática para el control institucional de medios informáticos, ofimáticos y de comunicaciones. Materiales y Métodos: investigación de desarrollo e innovación tecnológica, realizada durante 2018 y 2019, en dos etapas: 1) trabajo de mesa, definición de objetivos y establecimiento de pre-requisitos; 2) desarrollo de la aplicación, siguiendo la política de utilización de software libre. Las pruebas de funcionamiento y la evaluación se realizaron en la empresa MEDICuba S.A. Resultados: se desarrolló y registró una aplicación para el Control Informatizado de Medios de Informática, Ofimática y Comunicaciones (CIMIOC), con interfaz web, base de datos centralizada y un diseño adaptable a varios tipos de dispositivos. Permite registrar cualquier recurso de este tipo, su historial de movimiento, el estado técnico, los programas de mantenimiento y salvas de información, y ofrece diferentes reportes estadísticos. Conclusiones: la herramienta informática CIMIOC ofrece una solución robusta y económica para la gestión de las Tecnologías de la Información y las Comunicaciones, aplicable a cualquier institución. Facilita de manera objetiva el establecimiento de políticas que tributen a elevar los niveles de calidad en los servicios asociados a la utilización intensiva de estas tecnologías. Se recomienda generalizar la implantación en el Sistema Nacional de Salud.

Introduction: in the institutions belonging to the Ministry of Public Health of Cuba, the usual way of recording the control of Information and Communication Technologies has been through Excel sheets, word processors and printed technical sheets. Computerized control offers advantages, but existing computer applications worldwide are expensive or not adaptable to the requirements. Objective: to develop a computer tool for the institutional control of computer, office and communications media. Materials and Methods: technological development and innovation research, carried out during 2018 and 2019, in 2 stages: 1) table work, definition of objectives and establishment of pre-requisites. 2) development of the application, following the policy of use of free software. The performance tests and the evaluation were carried out in the company MEDICuba S.A. Results: an application for the Computerized Control of Informatics, Office Automation and Communications Media (CIMIOC) was developed and registered, with a web interface, centralized database and a design adaptable to various types of devices. It allows recording any resource of this type, its movement history, technical status, maintenance programs and information saves, and offers different statistical reports. Conclusions: the CIMIOC computer tool offers a robust and economical solution for ICT management, applicable to any institution. It objectively facilitates the establishment of policies that contribute to raising quality levels in services associated with the intensive use of ICT. Authors recommend to generalize the implementation in the National Health System.

Selection and validation of novel stable reference genes for qPCR analysis in EMT and MET.

Quantitative real-time polymerase chain reaction is a powerful tool for quantifying gene expression. The relative quantification relies on normalizing the data to reference genes or internal controls not modulated by the experimental conditions. The most widely used internal controls occasionally show changed expression patterns in different experimental settings, such as the mesenchymal to epithelial transition. Thus, identifying appropriate internal controls is of utmost importance. We analyzed multiple RNA-Seq datasets using a combination of statistical approaches such as percent relative range and coefficient of variance to define a list of candidate internal control genes, which was then validated experimentally and by using in silico analyses as well. We identified a group of genes as strong internal control candidates with high stability compared to the classical ones. We also presented evidence for the superiority of the percent relative range method for calculating expression stability in data sets with larger sample sizes. We used multiple methods to analyze data collected from several RNA-Seq datasets; we identified Rbm17 and Katna1 as the most stable reference genes in EMT/MET studies. The percent relative range approach surpasses other methods when analyzing datasets of larger sample sizes.

Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction.

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via qPCR analysis. Five miRNAs, bta-miR-29a, bta-miR-200a, bta-miR-26b, hsa-miR-27b-3p, and hsa-miR-30b-5p, were selected by microarray analyses, and their cycle threshold (Ct) values were further evaluated mathematically using geNorm, NormFinder, BestKeeper, and ∆Ct algorithms. The results revealed that both the miRNeasy Micro Kit and Maxwell RSC miRNA Tissue Kit are useful for the efficient recovery of RNA from bovine milk sEVs. According to the final stability ranking analyzed by RefFinder, hsa-miR-27b-3p and bta-miR-29a can be used as suitable internal control miRNAs in bovine milk sEVs. The study also indicated that using a suitable internal control miRNA may improve the reliability and accuracy of the qPCR analysis for normalization in bovine milk sEVs. To the best of our knowledge, this is the first study to uncover the suitable internal control miRNAs in bovine milk sEVs.

Selection of suitable internal control gene for assaying gene expression in rice through qRT-PCR during sheath blight infection.

qRT-PCR is a globally accepted technique for assaying gene expression in relative terms which compares the difference between critical threshold (CT) values of a gene calculated form two independently isolated RNA samples. Independent RNA isolations, however, include error due to batch effect which must be normalized for error-free calculation of relative gene expression. Hence, CT values of internal control (IC) genes are used for normalization during the calculation of expression fold-change in gene expression analysis. The expression of ICs genes expected to be stable in all the experimental conditions. However, it is almost impossible to find such a gene which do not depict expression fluctuation in response to the changes in experimental conditions. Hence, it is necessary to identify suitable IC gene(s) for any given experimental condition before conducting any particular gene expression study. Here, we examined the suitability of eight candidate IC genes, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic elongation factor-1 (eEF-1α), 25 S rRNA (25 S), 18 S rRNA (18 S), ubiquitin C E2 ligase (UBC), Actin (Act), ubiquitin 5 (UBQ5) and ubiquitin 10 (UBQ10), for assaying gene expression in rice during sheath blight infection. Our analysis suggest that GAPDH might be the IC of choice when expression studies include contrasting genotypes differing in their tolerance to sheath blight pathogen as well as progressive infection time. While if expression analysis have to be performed only in one genotype but under progressive sheath blight infection, UBQ5 might be chosen as IC because of its high expression stability under the proposed experimental setup.