Endogenous Reference Genes Research Articles

Circulating miR-323-3p as a novel potential plasma biomarker for multimorbidity burden and cognitive decline in middle-aged and older adults: results from the National Institute for Longevity Sciences–Longitudinal Study of Aging in Japan

BackgroundSeveral circulating microRNAs (miRNAs) in the blood are indicators of chronic diseases, but their association with multimorbidity burden is unknown. The aim of this study was to assess the association of plasma levels of circulating miR-323-3p and miR-135-3p with age, cognitive performance, and number of comorbidities in middle-aged and older individuals. MethodsData from 295 community dwellers (≥40 years) who participated in the second wave (2000‒2002) of the National Institute for Longevity Sciences-Longitudinal Study of Aging in Japan were analyzed. miRNAs were isolated from the plasma, and miR-323-3p and miR-135-3p levels were measured using quantitative reverse-transcription polymerase chain reaction and normalized, with miR-16 as an endogenous reference gene. Cognitive performance was assessed using the Information and Similarities subtests and the Digit Symbol Substitution Test of the Wechsler Adult Intelligence Scale-Revised Short Form (WAIS-R-SF). Correlation tests and multivariate general linear regression analyses were performed to examine the relationship among circulating miRNA levels, burden of multimorbidity, and cognitive performance. ResultsThe mean age of the participants was 59.1 ± 10.3 years. Pearson's correlation analysis revealed that the miR-323-3p level positively correlated with age and number of comorbidities but negatively correlated with WAIS-R-SF subtest performance. In men, miR-323-3p level negatively correlated with the performance of all three WAIS-R-SF subtests. The general linear regression analysis showed that the miR-323-3p level increased in participants with four comorbidities compared with those with one comorbidity. ConclusionCirculating miR-323-3p level is associated with the burden of multimorbidity and decreased cognitive performance in middle-aged and older adults.

COMPARISON OF YIELD AND QUALITY OF CIRCULATING CELL-FREE DNA FROM K2EDTA AND K3EDTA COLLECTIONS IN HEALTHY SUBJECTS

Background: Circulating cell-free DNA (cfDNA) is a biomarker for various clinical applications, including detecting and monitoring cancer. However, blood collection tubes can affect the yield and quality of cfDNA. Since specific cfDNA collection tubes are costly, K2EDTA and K3EDTA anticoagulant tubes are alternatives in routine clinical laboratories. Objectives: This study aimed to compare the efficiency of cfDNA extraction from plasma collected in K2EDTA and K3EDTA tubes and evaluate implementation for molecular diagnostics. Methods: Blood samples from 38 healthy subjects were collected in K2EDTA and K3EDTA tubes that were processed within 2 hours. The extracted cfDNA was measured and performed using SYBR Green-based qPCR for three endogenous reference genes (GAPDH, HPRT1, TFRC). The cfDNA yield and the amplification efficiency of these genes were compared between K2EDTA and K3EDTA tubes using the Mann-Whitney U test. Results: There were no significant differences in cfDNA concentration between K2EDTA and K3EDTA tubes (p=0.051). However, qPCR analysis revealed significantly higher copy numbers of TFRC and HPRT1 in K2EDTA tubes than in K3EDTA tubes (p<0.05). No significant difference was found for GAPDH. Conclusion: The results indicate that K2EDTA and K3EDTA tubes are an alternative option for cfDNA analysis if samples are processed quickly after a blood draw, which offers flexibility and cost savings in resource-limited areas.

A portable multifunctional biosensor based on a newly screened endogenous reference gene for pork adulteration detection

The adulteration of animal-derived food is a widespread problem with meat adulteration emerging as a global concern. To address the need for on-site rapid detection and visual analysis, a portable multifunction biosensor (PMB) was developed. This device integrates Recombinase Polymerase Amplification (RPA), CRISPR/Cas12a assay, and a custom-designed 3D-printed multifunction device, specifically for the visual and rapid detection of pork in food. Initially, a screening model was constructed using Perl bioinformatics based on chromosomal gene information, and the LOC110262058 gene was selected as a reliable endogenous reference gene for pigs. Subsequently, a one-tube RPA-CRISPR/Cas12a system was established to simplify the procedures, converting pork-specific signals into fluorescence while preventing cross-contamination. In addition, an independently designed 3D-printed multifunction device was developed to support the RPA-CRISPR/Cas12a system for one-tube detection, incorporating functions such as heating, centrifugation, and imaging. The reaction conditions of the PMB were optimized to reduce the reaction time to 20–30 min with a sensitivity of 0.01 g/100 g. The analysis of actual samples indicated that the PMB has high selectivity and practical effectiveness for pork detection. The proposed biosensor offers several detection advantages, including output, ease of use, stability, and independence from complex equipment, making it a powerful method for rapid pork detection in food.

Ces44T as an endogenous reference gene in real-time quantitative PCR detection of tiger nut (Cyperus esculentus) ingredients in food

Tiger nut (Cyperus esculentus) is a highly adaptable and nutritious plant that can be used to produce health-enhancing food products, edible oil, and functional ingredients. To ensure transparency, reliability, and accurate labeling throughout the food supply chain, accurate methods for identifying food ingredients are indispensable. In this study, the Ces44T gene was identified and validated as the endogenous reference gene for tiger nut. A real-time quantitative PCR assay targeting the Ces44T gene was developed, demonstrating exceptional specificity in distinguishing tiger nut from other plant species, and exhibiting remarkable stability across different tiger nut cultivars. Standard curves were established to validate the reliability and repeatability of the assay, displaying good linearity (R2 > 0.99) and PCR efficiency (90–110 %). The assay was estimated to have a limit of detection (LOD) of 0.0033 ng of tiger nut genomic DNA per reaction and a limit of quantification (LOQ) of 0.013 ng. We applied this assay to both laboratory-prepared samples and commercial food products, confirming its excellent specificity and robust performance. In conclusion, the developed real-time quantitative PCR assay provides an accurate means for the identification and quantification of tiger nut ingredients in various samples.

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

GMOIT: a tool for effective screening of genetically modified crops

BackgroundAdvancement in agricultural biotechnology has resulted in increasing numbers of commercial varieties of genetically modified (GM) crops worldwide. Though several databases on GM crops are available, these databases generally focus on collecting and providing information on transgenic crops rather than on screening strategies. To overcome this, we constructed a novel tool named, Genetically Modified Organisms Identification Tool (GMOIT), designed to integrate basic and genetic information on genetic modification events and detection methods.ResultsAt present, data for each element from 118 independent genetic modification events in soybean, maize, canola, and rice were included in the database. Particularly, GMOIT allows users to customize assay ranges and thus obtain the corresponding optimized screening strategies using common elements or specific locations as the detection targets with high flexibility. Using the 118 genetic modification events currently included in GMOIT as the range and algorithm selection results, a “6 + 4” protocol (six exogenous elements and four endogenous reference genes as the detection targets) covering 108 events for the four crops was established. Plasmids pGMOIT-1 and pGMOIT-2 were constructed as positive controls or calibrators in qualitative and quantitative transgene detection.ConclusionsOur study provides a simple, practical tool for selecting, detecting, and screening strategies for a sustainable and efficient application of genetic modification.

Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria

The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection’ with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

Determination of Appropriate Endogenous Reference Genes for RT-qPCR Analysis in Syrian (Golden) Hamsters and Mongolian Gerbils

Purpose: The use of hamsters and gerbils has increased significantly in a variety of fields, including biological rhythms, reproductive biology, immunology, oncology, and many others. Material and Methods: The most stable genes in Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus) were assessed using 32 reference genes for normalization in RT-qPCR analysis. Adrenal, cerebral cortex, heart, hypothalamus, kidney, liver, lung and testis tissues were used to extract and purify RNAs. GeNorm was used to determine the gene expression stabilities of 14 candidate endogenous genes from each tissue that was compatible for both animals. Results: Under our experimental conditions, we discovered that two endogenous genes are adequate for each tissue to perform RT-qPCR normalization. There were differences in the most stable genes between species and tissues. Conclusion: We suggest that combinations of endogenous genes ought to be carefully chosen under various experimental circumstances.

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods.

Endogenous reference genes play a crucial role in the qualitative and quantitative PCR detection of genetically modified crops. Currently, there are no systematic studies on the banana endogenous reference gene. In this study, the MaSPS1 gene was identified as a candidate gene through bioinformatics analysis. The conservation of this gene in different genotypes of banana was tested using PCR, and its specificity in various crops and fruits was also examined. Southern blot analysis showed that there is only one copy of MaSPS1 in banana. The limit of detection (LOD) test showed that the LOD of the conventional PCR method is approximately 20 copies. The real-time fluorescence quantitative PCR (qPCR) method also exhibited high specificity, with a LOD of approximately 10 copies. The standard curve of the qPCR method met the quantitative requirements, with a limit of quantification (LOQ) of 1.14 × 10-2 ng-about 20 copies. Also, the qPCR method demonstrated good repeatability and stability. Hence, the above results indicate that the detection method established in this study has strong specificity, a low detection limit, and good stability. It provides a reliable qualitative and quantitative detection system for banana.

Hprt Serves as an Ideal Reference Gene for qRT-PCR Normalization in Rat DRG Neurons.

To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons. The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression. All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA (Rn18s) and β-actin (Actb) exhibited the most significant changes, while ubiquitin C (Ubc), hypoxanthine phosphoribosyl transferase (Hprt), and mitochondrial ribosomal protein L10 (Mrpl10) showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, Actb was found to be the most unstable reference gene, while Hprt was the relatively most stable reference gene. The use of unstable reference genes Actb and ankyrin repeat domain 27 (Ankrd27) as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene Hprt had small inter-assay variation and high stability. Our observations indicate that Hprt is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.

An efficient and accurate droplet digital PCR method for rapid transgene copy number detection and homozygous identification in cotton (Gossypium hirsutum)

Transgene copy number characterization and homozygous identification are important steps for both the scientific research and transgenic breeding in plant. Traditionally, Southern blotting, quantitative PCR, genome sequencing and genetic analysis are the major methods to achieve these objectives. These methods are laborious, time-consuming and cost-expensive. Digital PCR is a breakthrough technology that is able to provide sensitive and absolute nucleic acid quantification. However, its application is rarely studied in transgene copy number detection and homozygous identification, especially in cotton, an important industrial crop worldwide. Here, we developed a droplet digital PCR (ddPCR) system to rapidly and efficiently detect the transgene copy number in cotton. The GhTPS, a single gene in per subgenome of Gossypium hirsutum, and NPT II, a marker gene in the transgenic T-DNA, were selected as the endogenous reference gene and exogenous insertion sequences, respectively. Results showed that probe concentration at 150 nM and DNA concentration at 5.0 ng/µL were the best conditions to perform ddPCR in tetraploid cotton. The detected T-DNA copy numbers obtained from the developed ddPCR system were in consistent with the results from Southern blotting analysis and genome resequencing results, respectively, demonstrating that the developed ddPCR system is accurate and efficient in identifying the T-DNA copy number. Moreover, the homozygous and heterozygous T1 offspring plants from a T0 plant were efficiently distinguished using this approach, greatly shortening the selection cycles of homozygous plants. The ddPCR system was further applied to detect the transgenic Bt copy numbers in commercialized cotton varieties, indicating the suitability and convenience in identifying transgenic cotton with different origins. This study provides a novel, rapid and efficient method for copy number and homozygous determination in transgenic cotton.

Evaluation of a Porcine Endogenous Reference Gene (Internal Sample Control) in a Porcine Reproductive and Respiratory Syndrome Virus RT-qPCR.

Endogenous reference genes are used in gene-expression studies to "normalize" the results and, increasingly, as internal sample controls (ISC) in diagnostic quantitative polymerase chain reaction (qPCR). Three studies were conducted to evaluate the performance of a porcine-specific ISC in a commercial porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription-qPCR. Study 1 evaluated the species specificity of the ISC by testing serum from seven non-porcine domestic species (n = 34). In Study 2, the constancy of ISC detection over time (≥42 days) was assessed in oral fluid (n = 130), serum (n = 215), and feces (n = 132) collected from individual pigs of known PRRSV status. In Study 3, serum (n = 150), oral fluid (n = 150), and fecal samples (n = 75 feces, 75 fecal swabs) from commercial herds were used to establish ISC reference limits. Study 1 showed that the ISC was porcine-specific, i.e., all samples from non-porcine species were ISC negative (n = 34). In Study 2, the ISC was detected in all oral fluid, serum, and fecal samples, but differed in concentration between specimens (p < 0.05; mixed-effects regression model). The results of Study 3 were used to establish ISC reference limits for the 5th, 2.5th and 1.25th percentiles. Overall, the ISC response was consistent to the point that failure in detection is sufficient justification for re-testing and/or re-sampling.

High Quality Rna Isolation and Exogenous Reference Gene for Real-time Pcr In Cedrela Odorata Seed Physiology Studies

Seed banks represent an important strategy for the conservation of forest genetic resources, although a basic understanding of the physiological changes that seeds undergo during storage that affect quality and germination is still lacking for most tropical and subtropical species. Here, we describe the optimisation of an RNA isolation procedure and reference gene normalisation for expression analysis in Cedrela odorata (cedro or Spanish cedar) seeds during different physiological states, as well as in the steady-state stem and leaf. The expression profiles of five endogenous candidate reference genes ( 18S , EF1α , GAPDH , CDC27B , PP2A2 ) and an exogenous ( HMBS ) gene were evaluated by using dedicated algorithms, including Genorm, Normfinder, Bestkeeper and Ct. We found that the expression of all endogenous genes varied considerably in response to both ageing and hydration. Therefore, using the external HMBS was a suitable alternative to evaluate gene expression in these highly contrasting physiological conditions. The reference genes EF1α and GAPDH were the most stable, and could be used for normalisation of qRT-PCR results under specific circumstances.

Quantitative evaluation of endogenous reference genes for ddPCR under salt stress using a moderate halophile.

Droplet digital PCR (ddPCR) is being increasingly adopted for gene detection and quantification because of its higher sensitivity and specificity. According to previous observations and our laboratory data, it is essential to use endogenous reference genes (RGs) when investigating gene expression at the mRNA level under salt stress. This study aimed to select and validate suitable RGs for gene expression under salt stress using ddPCR. Six candidate RGs were selected based on the tandem mass tag (TMT)-labeled quantitative proteomics of Alkalicoccus halolimnae at four salinities. The expression stability of these candidate genes was evaluated using statistical algorithms (geNorm, NormFinder, BestKeeper and RefFinder). There was a small fluctuation in the cycle threshold (Ct) value and copy number of the pdp gene. Its expression stability was ranked in the vanguard of all algorithms and was the most suitable RG for quantification of expression by both qPCR and ddPCR of A. halolimnae under salt stress. Single RG pdp and RG combinations were used to normalize the expression of ectA, ectB, ectC and ectD under four salinities. The present study constitutes the first systematic analysis of endogenous RG selection for halophiles responding to salt stress. This work provides a valuable theory and an approach reference of internal control identification for ddPCR-based stress response models.

Screening and validating of endogenous reference genes in Chlorella sp. TLD 6B under abiotic stress

Chlorella sp. TLD 6B, a microalgae growing in the Taklamakan Desert, Xinjiang of China, is a good model material for studying the physiological and environmental adaptation mechanisms of plants in their arid habitats, as its adaptation to the harsh desert environment has led to its strong resistance. However, when using real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the gene expression of this algae under abiotic stress, it is essential to find the suitable endogenous reference genes so to obtain reliable results. This study assessed the expression stability of 9 endogenous reference genes of Chlorella sp. TLD 6B under four abiotic stresses (drought, salt, cold and heat). These genes were selected based on the analysis results calculated by the three algorithmic procedures of geNorm, NormFinder, and BestKeeper, which were ranked by refinder. Our research showed that 18S and GTP under drought stress, 18S and IDH under salt stress, CYP and 18S under cold stress, GTP and IDH under heat stress were the most stable endogenous reference genes. Moreover, UBC and 18S were the most suitable endogenous reference gene combinations for all samples. In contrast, GAPDH and α-TUB were the two least stable endogenous reference genes in all experimental samples. Additionally, the selected genes have been verified to be durable and reliable by detecting POD and PXG3 genes using above endogenous reference genes. The identification of reliable endogenous reference genes guarantees more accurate RT-qPCR quantification for Chlorella sp. TLD 6B, facilitating functional genomics studies of deserts Chlorella as well as the mining of resistance genes.

Screening and evaluation of endogenous reference genes for miRNA expression analysis in forensic body fluid samples

MicroRNA (miRNA)-based methods for body fluid identification are promising tools in the practice of forensic science. The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. Then, the expression levels of 10 relatively stable candidate reference RNAs in 100 samples were determined and further analysed using four commonly employed programs (geNorm, NormFinder, BestKeeper and ΔCq). According to the comprehensive stability rankings of the four algorithms, miR-320a-3p was validated as the most stable endogenous reference gene among the five forensically relevant body fluids, followed by miR-484, SNORD43, miR-320c and RNU6b. Moreover, the combined application of miR-320a-3p with RNU6b could increase the normalization effect. In addition, a total of 56 mock samples placed outdoors and indoors for different times were prepared to further evaluate the stability of candidate reference RNAs, and miR-320a-3p remained the preferred reference gene. Furthermore, the relative expression levels of publicly accepted body fluid-specific miRNAs were determined in 30 samples to verify the practicality and effectiveness of the reference genes. Our results revealed a set of alternative reference genes and could promote the development and application of miRNA-based body fluid identification by determining optional reference genes for strict normalization.

Identification of RNU44 as an Endogenous Reference Gene for Normalizing Cell-Free RNA in Tuberculosis.

Normalization of cell-free RNA (cf-RNA) concentration can be affected by variable experimental conditions and thus impact the performance of their diagnostic potential. Our study aimed to identify appropriate endogenous reference genes for cf-RNA biomarker evaluation in the diagnosis of tuberculosis (TB). Subjects consisting of patients with TB with and without malignancy, patients with pneumonia, and healthy controls were recruited. Candidate reference genes were screened and identified by literature reviewing and RNA-Seq analysis. Expression levels of the candidate genes were determined by reverse-transcription real-time quantitative polymerase chain reaction in plasma from patients with TB and healthy controls. The stability of gene expression was assessed by geNorm, NormFinder, BestKeeper, the Comparative Delta Ct method, and RefFinder. Differential expression of 2 small RNAs (sRNAs) encoding by genome of Mycobacterium tuberculosis in plasma of patients with TB were determined by both absolute quantification and relative quantification with candidate reference genes. According to the stability ranking analyzed with the 5 computational programs, the top 4 candidates-miR-93, RNU44, miR-16, and glyceraldehyde 3-phosphate dehydrogenase-were used to normalize the transcript levels of 2 mycobacterial sRNAs, MTS2823 and MTS1338, which were observed to have higher copy numbers in the plasma of patients with TB. Normalization with RNU44 displayed significantly higher levels of the 2 M tuberculosis sRNAs in the patients' plasma than those of healthy controls. RNU44 was demonstrated as a proper endogenous gene for cf-RNA normalization in TB diagnosis.

Circulating tumour cell combined with DNA methylation for early detection of hepatocellular carcinoma.

Background: The inadequate early detection strategies makes hepatocellular carcinoma (HCC) patients with poor prognisis. Therefore, more effective detection methods are urgently needed for early detection and early intervention of HCC. Methods: 17 cases of suspected HCC patients and 11 cases of HBV-related decompensated cirrhosis (HBV-DeCi) patients were enrolled. For each patient, 5ml blood sample was separated into circulating tumor cells (CTCs) and plasma, CTCs were stained with Diff staining for counting. Plasma was used for extracting cell free DNA (cfDNA) and then analyzed by qMSP assay. Ct values were recorded for GNB4 and Riplet as target genes and β-actin as an endogenous reference gene. Finally, clinical efficacy of CTC count combined with GNB4/Riplet methylation detection for early diagnosis of HCC was analyzed. Results: The CTC of HCC patients has pleomorphic characteristics, but it is difficult to distinguish from other blood cells with non-obviously pleomorphic of CTC. Although a small number of CTCs can also be detected in HBV-DeCi patients (control group), the number is significantly lower than that in HCC patients, the sensitivity and specificity of CTC for HCC detection were 70.6% and 90.9% (AUC = 0.81). The Ct values of GNB4 and Riplet methylation were significantly different between HCC patients and control group patients. When CTC combined with two genes, the AUC value was significantly increased to 0.98, the sensitivity was 88.2%, and the specificity was 100%. Conclusion: Our study has developed a novel test that CTC count combined with GNB4/Riplet methylation detection and showed its high performance for early diagnosis of HCC.

Identification of s9ap used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of Pleurotus eryngii.

Pleurotus eryngii is a kind of edible fungi with good quality, and it is popular among consumers. At present, some adulterated edible fungi are available in the market. The rights and interests of consumers can be ensured by establishing a practical edible fungi detection system. Among the existing methods for detecting food adulteration, endogenous reference gene amplification is convenient and reliable. However, no ideal endogenous reference gene is available for P. eryngii. In this study, s9ap was screened as an endogenous reference gene through sequence alignment. Qualitative and quantitative PCR analysis of this gene was carried out in one P. eryngii variety and 18 other species. The detection limit of quantitative PCR was 400 pg, and no s9ap amplification products were detected in the 18 other species. This study confirmed that s9ap was an ideal endogenous reference gene for the detection of P. eryngii. This method was also suitable for processed food products.

Internal reference genes with the potential for normalizing quantitative PCR results for oral fluid specimens.

In basic research, testing of oral fluid specimens by real-time quantitative polymerase chain reaction (qPCR) has been used to evaluate changes in gene expression levels following experimental treatments. In diagnostic medicine, qPCR has been used to detect DNA/RNA transcripts indicative of bacterial or viral infections. Normalization of qPCR using endogenous and exogenous reference genes is a well-established strategy for ensuring result comparability by controlling sample-to-sample variation introduced during sampling, storage, and qPCR testing. In this review, the majority of recent publications in human (n = 136) and veterinary (n = 179) medicine did not describe the use of internal reference genes in qPCRs for oral fluid specimens (52.9% animal studies; 57.0% human studies). However, the use of endogenous reference genes has not been fully explored or validated for oral fluid specimens. The lack of valid internal reference genes inherent to the oral fluid matrix will continue to hamper the reliability, reproducibility, and generalizability of oral fluid qPCR assays until this issue is addressed.