Fundamental behaviors emerge from simulations of a living minimal cell.

We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

Prevalence and Predictors of Oral Treponema pallidum Detection by Quantitative Polymerase Chain Reaction in Early Syphilis.

BACKGROUND: Treponema pallidum prevalence and burden at oral and lesion sites in adults with early syphilis were assessed by quantitative polymerase chain reaction (qPCR). Factors associated with oral shedding were also examined. METHODS: Pretreatment oral and lesion swabs were collected from adults with early syphilis in a US multicenter syphilis treatment trial. Oral swabs were collected in the presence and absence of oral lesions. Following DNA extraction, qPCR and whole-genome sequencing (WGS) were performed to assess burden and strain variability. RESULTS: All 32 participants were male, mean age was 35 years, and 90.6% with human immunodeficiency virus (HIV). T. pallidum oral PCR positivity varied by stage: 16.7% primary, 44.4% secondary, and 62.5% in early latent syphilis. Median oral T. pallidum burden was highest in secondary syphilis at 63.2 copies/µL. Lesion PCR positivity was similar in primary (40.0%) and secondary syphilis (38.5%). Age 18-29 years was significantly associated with oral shedding (vs age 40+ years) in adjusted models. WGS identified 2 distinct strains. CONCLUSIONS: T. pallidum DNA was directly detected at oral and lesion sites in a significant proportion of men with early syphilis. Younger age was associated with oral shedding. Ease of oral specimen collection and increased PCR availability suggest opportunities to improve syphilis diagnostic testing. Clinical Trials Registration. NCT03637660.

A novel qPCR-based technique for identifying avian sex: An illustration within embryonic craniofacial bone.

Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.

Semi-quantitative group testing for efficient and accurate qPCR screening of pathogens with a wide range of loads.

BACKGROUND: Pathogenic infections pose a significant threat to global health, affecting millions of people every year and presenting substantial challenges to healthcare systems worldwide. Efficient and timely testing plays a critical role in disease control and transmission prevention. Group testing is a well-established method for reducing the number of tests needed to screen large populations when the disease prevalence is low. However, it does not fully utilize the quantitative information provided by qPCR methods, nor is it able to accommodate a wide range of pathogen loads. RESULTS: To address these issues, we introduce a novel adaptive semi-quantitative group testing (SQGT) scheme to efficiently screen populations via two-stage qPCR testing. The SQGT method quantizes cycle threshold (Ct) values into multiple bins, leveraging the information from the first stage of screening to improve the detection sensitivity. Dynamic Ct threshold adjustments mitigate dilution effects and enhance test accuracy. Comparisons with traditional binary outcome GT methods show that SQGT reduces the number of tests by 24% on the only complete real-world qPCR group testing dataset from Israel, while maintaining a negligible false negative rate. CONCLUSION: In conclusion, our adaptive SQGT approach, utilizing qPCR data and dynamic threshold adjustments, offers a promising solution for efficient population screening. With a reduction in the number of tests and minimal false negatives, SQGT holds potential to enhance disease control and testing strategies on a global scale.

The effect of the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy and its relationship with toxoplasmosis.

This study aims to investigate the relationship between toxoplasmosis and this pathway, which may be effective in the formation of epilepsy by acting through the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy. In the study, four different experimental groups were formed by selecting Toxoplasma gondii IgG positive and negative patients with idiopathic epilepsy and healthy controls. Experimental groups were as follows: Group 1: Epilepsy+/Toxo- (E+, T-) (n = 10), Group 2: Epilepsy-/Toxo- (E-, T-) (n = 10), Group 3: Epilepsy-/Toxo+ (E-, T+) (n = 10), Group 4: Epilepsy+/Toxo+ (E+, T+) (n = 10). HMGB1, RAGE, TLR4, TLR1, TLR2, TLR3, IRAK1, IRAK2, IKBKB, IKBKG, BCL3, IL1ß, IL10, 1 L8 and TNFα mRNA expression levels in the HMGB/RAGE/TLR4/NF-κB signalling pathway were determined by quantitative simultaneous PCR (qRT-PCR) after collecting blood samples from all patients in the groups. Statistical analysis was performed by one-way ANOVA followed by LSD post-hoc tests, and p < 0.05 was considered to denote statistical significance. The gene expression levels of HMGB1, TLR4, IL10, IL1B, IL8, and TLR2 were significantly higher in the G1 group than in the other groups (p < 0.05). In the G3 group, RAGE and BCL3 gene expression levels were significantly higher than in the other groups (p < 0.05). In the G4 group, however, IRAK2, IKBKB, and IKBKG gene expression levels were significantly higher than in the other groups (p < 0.05). HMGB1, TLR4, IRAK2, IKBKB, IL10, IL1B, IL1B, and IL8 in this signalling pathway are highly expressed in epilepsy patients in G1 and seizures occur with the stimulation of excitatory mechanisms by acting through this pathway. The signalling pathway in epilepsy may be activated by HMGB1, TLR4, and TLR2, which are considered to increase the level of proinflammatory cytokines. In T. gondii, this pathway is activated by RAGE and BCL3.

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR.

BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

ExonSurfer: a web-tool to design primers at exon-exon junctions.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. RESULTS: ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. CONCLUSION: ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

DYT-THAP1: exploring gene expression in fibroblasts for potential biomarker discovery.

Dystonia due to pathogenic variants in the THAP1 gene (DYT-THAP1) shows variable expressivity and reduced penetrance of ~ 50%. Since THAP1 encodes a transcription factor, modifiers influencing this variability likely operate at the gene expression level. This study aimed to assess the transferability of differentially expressed genes (DEGs) in neuronal cells related to pathogenic variants in the THAP1 gene, which were previously identified by transcriptome analyses. For this, we performed quantitative (qPCR) and Digital PCR (dPCR) in cultured fibroblasts. RNA was extracted from THAP1 manifesting (MMCs) and non-manifesting mutation carriers (NMCs) as well as from healthy controls. The expression profiles of ten of 14 known neuronal DEGs demonstrated differences in fibroblasts between these three groups. This included transcription factors and targets (ATF4, CLN3, EIF2A, RRM1, YY1), genes involved in G protein-coupled receptor signaling (BDKRB2, LPAR1), and a gene linked to apoptosis and DNA replication/repair (CRADD), which all showed higher expression levels in MMCs and NMCs than in controls. Moreover, the analysis of genes linked to neurological disorders (STXBP1, TOR1A) unveiled differences in expression patterns between MMCs and controls. Notably, the genes CUEDC2, DRD4, ECH1, and SIX2 were not statistically significantly differentially expressed in fibroblast cultures. With > 70% of the tested genes being DEGs also in fibroblasts, fibroblasts seem to be a suitable model for DYT-THAP1 research despite some restrictions. Furthermore, at least some of these DEGs may potentially also serve as biomarkers of DYT-THAP1 and influence its penetrance and expressivity.

Prenatal tetrahydrocannabinol and cannabidiol exposure produce sex-specific pathophysiological phenotypes in the adolescent prefrontal cortex and hippocampus.

Clinical and preclinical evidence has demonstrated an increased risk for neuropsychiatric disorders following prenatal cannabinoid exposure. However, given the phytochemical complexity of cannabis, there is a need to understand how specific components of cannabis may contribute to these neurodevelopmental risks later in life. To investigate this, a rat model of prenatal cannabinoid exposure was utilized to examine the impacts of specific cannabis constituents (Δ9-tetrahydrocannabinol [THC]; cannabidiol [CBD]) alone and in combination on future neuropsychiatric liability in male and female offspring. Prenatal THC and CBD exposure were associated with low birth weight. At adolescence, offspring displayed sex-specific behavioural changes in anxiety, temporal order and social cognition, and sensorimotor gating. These phenotypes were associated with sex and treatment-specific neuronal and gene transcriptional alterations in the prefrontal cortex, and ventral hippocampus, regions where the endocannabinoid system is implicated in affective and cognitive development. Electrophysiology and RT-qPCR analysis in these regions implicated dysregulation of the endocannabinoid system and balance of excitatory and inhibitory signalling in the developmental consequences of prenatal cannabinoids. These findings reveal critical insights into how specific cannabinoids can differentially impact the developing fetal brains of males and females to enhance subsequent neuropsychiatric risk.

Encephalitozoon cuniculi Microsporidia in Cerebrospinal Fluid from Immunocompetent Patients, Czech Republic.

We retrospectively analyzed of 211 frozen cerebrospinal fluid samples from immunocompetent persons in the Czech Republic and detected 6 Encephalitozoon cuniculi-positive samples. Microsporidiosis is generally underestimated and patients are not usually tested for microsporidia, but latent infection in immunodeficient and immunocompetent patients can cause serious complications if not detected and treated.

Large-Scale Outbreak of Mycoplasma pneumoniae Infection, Marseille, France, 2023-2024.

We report a large-scale outbreak of Mycoplasma pneumoniae respiratory infections encompassing 218 cases (0.8% of 26,449 patients tested) during 2023-2024 in Marseille, France. The bacterium is currently circulating and primarily affects children <15 years of age. High prevalence of co-infections warrants the use of a syndromic diagnostic strategy.

Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR.

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Development of a multigenomic liquid biopsy (PROSTest) for prostate cancer in whole blood.

INTRODUCTION: We describe the development of a molecular assay from publicly available tumor tissue mRNA databases using machine learning and present preliminary evidence of functionality as a diagnostic and monitoring tool for prostate cancer (PCa) in whole blood. MATERIALS AND METHODS: We assessed 1055 PCas (public microarray data sets) to identify putative mRNA biomarkers. Specificity was confirmed against 32 different solid and hematological cancers from The Cancer Genome Atlas (n = 10,990). This defined a 27-gene panel which was validated by qPCR in 50 histologically confirmed PCa surgical specimens and matched blood. An ensemble classifier (Random Forest, Support Vector Machines, XGBoost) was trained in age-matched PCas (n = 294), and in 72 controls and 64 BPH. Classifier performance was validated in two independent sets (n = 263 PCas; n = 99 controls). We assessed the panel as a postoperative disease monitor in a radical prostatectomy cohort (RPC: n = 47). RESULTS: A PCa-specific 27-gene panel was identified. Matched blood and tumor gene expression levels were concordant (r = 0.72, p < 0.0001). The ensemble classifier ("PROSTest") was scaled 0%-100% and the industry-standard operating point of ≥50% used to define a PCa. Using this, the PROSTest exhibited an 85% sensitivity and 95% specificity for PCa versus controls. In two independent sets, the metrics were 92%-95% sensitivity and 100% specificity. In the RPCs (n = 47), PROSTest scores decreased from 72% ± 7% to 33% ± 16% (p < 0.0001, Mann-Whitney test). PROSTest was 26% ± 8% in 37 with normal postoperative PSA levels (<0.1 ng/mL). In 10 with elevated postoperative PSA, PROSTest was 60% ± 4%. CONCLUSION: A 27-gene whole blood signature for PCa is concordant with tissue mRNA levels. Measuring blood expression provides a minimally invasive genomic tool that may facilitate prostate cancer management.

A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Influence of storage solution, temperature, assay time and concentration on RT-qPCR nucleic acid detection for SARS-CoV-2 detection of SARS-CoV-2 by the RT-qPCR.

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is an important method for the early diagnosis of coronavirus disease 2019 (COVID-19). This study investigated the effects of storage solution, temperature and detection time on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection by RT-qPCR. Various concentrations of SARS-CoV-2 were added to inactive and non-inactive storage solution and the viral suspensions were stored at various temperatures (room temperature, 4, -20 and -80 °C). Then, at five different detection time points, the Ct values were determined by RT-qPCR. Active and inactive storage solutions and storage temperature have a great impact on the detection of N gene of SARS-CoV-2 at different concentration corridors but have little impact on the ORF gene. The storage time has a greater impact on the N gene and ORF gene at high concentrations but has no effect on the two genes at low concentrations. In conclusion, storage temperature, storage time and storage status (inactivated, non-inactivated) have no effect on the nucleic acid detection of SARS-CoV-2 at the same concentration. For different concentrations of SARS-CoV-2, the detection of N gene is mainly affected.

Transcriptome-based identification and validation of reference genes for corm growth stages, different tissues, and drought stress in Taro (Colocasia esculenta).

Taro is a widely utilized starch resource plant. It is essential to quantify the expression levels of functional genes associated with taro growth using real-time quantitative polymerase chain reaction (RT-qPCR). However, to obtain reliable RT-qPCR results, appropriate reference genes (RGs) are required for data normalization. In this study, we screened seven novel candidate RGs using transcriptome datasets from taro, encompassing data from growth corms and various tissues. The expression stability of these seven new RGs, along with the commonly used RGs Actin, EF1-α, and ß-tubulin, was assessed using Delta Ct, BestKeeper, geNorm, and NormFinder algorithms. Furthermore, we conducted a comprehensive analysis using the RefFinder program and validated the results using the target gene, CeAGPL1. The findings revealed that ACY-1 and PIA2 were the optimal multiple RGs for normalization during corm growth, while COX10 and Armc8 were suitable for samples including various types of tissues. Furthermore, we found three RGs, Armc8, COX10 and CCX4L, were the optimal RGs for drought stress. This study assessed the suitability of RGs in taro for the first time. The identified RGs provide valuable resources for studying corm growth, diverse tissues, and drought stress. This study contributes to the advancement of our understanding of the underlying mechanisms that govern the growth of taro.