Reverse Transcriptase-quantitative Polymerase Chain Reaction Analysis Research Articles

By modulating miR-525-5p/Bax axis, LINC00659 promotes vascular endothelial cell apoptosis.

Deep vein thrombosis (DVT) is a vascular disease that has no effective treatment at present. Endothelial cells play a crucial role in the processes vasoconstriction, platelet activation, and blood coagulation and are an integral part of the vascular response to injury resulting in thrombus formation. The aim of this study was to investigate the roles and mechanisms of long noncoding RNA LINC00659 (LINC00659) in endothelial cells. The functions of LINC00659 and miR-525-5p on endothelial cells were explored by cell transfection assays, and the expression levels of LINC00659, miR-525-5p, and Bax in human umbilical vein endothelial cells(HUVECs) were assessed with reverse transcriptase-quantitative polymerase chain reaction(RT-qPCR). Binding sites of LINC00659 and miR-525-5p were subsequently analyzed with bioinformatics software, and validated with dual-luciferase reporter gene assay. Effects of LINC00659 and miR-525-5p on proliferation and apoptosis of HUVECs were detected with MTT (3-(45)-dimethylthiahiazo (-z-y1)-35-di-phenytetrazoliumromide) assay and flow cytometry. RT-qPCR and western blot analysis were used to evaluate the mRNA and protein levels of apoptosis-related markers Bcl-2 and Bax in HUVECs. LINC00659 directly targeted and negatively regulated miR-525-5p, and Bax was a target of miR-525-5p. Upregulation of LINC00659 could inhibit proliferation and promote apoptosis of HUVECs, while the silencing of LINC00659 could increase the viability of HUVECs and inhibit apoptosis via upregulating miR-525-5p. Further mechanistic studies revealed miR-525-5p could negatively regulate Bax in HUVECs, and increased the viability of HUVECs and inhibited apoptosis by downregulating Bax expression. LINC00659 played an important role in DVT by regulating the apoptosis of vascular endothelial cells through regulating miR-525-5p/Bax axis.

Development of a Standardized, DNA-Based Next Generation Sequencing Assay for Assessment of Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML) As the Primary Endpoint in the Agility Study

Background:NPM1 mutations (NPM1m) are a well-characterized biomarker for minimal residual disease (MRD) detection in acute myeloid leukemia (AML). Based on our retrospective meta-analysis across multiple trials and prior published studies, we are evaluating the utility of MRD complete response as a surrogate biomarker for clinical benefit in the AGILITY study (NCT05020665) to support a potential accelerated approval. Currently, measurement of NPM1m RNA transcripts (relative to ABL1) using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is considered the gold standard for monitoring of MRD; however, DNA-based next generation sequencing (NGS) provides several logistical and technical advantages over RT-qPCR in a global registrational trial setting. NGS allows for detection of all NPM1m variant alleles in a single assay (more closely approximating the variant allelic fraction [VAF] from leukemic cells vs NPM1m transcripts). Furthermore, longer stability of DNA during transit alleviates logistical challenges related to sample processing across different labs and transport from sites worldwide to a central testing site. Purpose: To develop a standardized and highly sensitive DNA-based NGS assay for detection of MRD in NPM1m AML patients at the end of 2 cycles of chemotherapy in the AGILITY study and to assess its performance against an established RT-qPCR method. Methods: Assay performance was assessed by serially diluting NPM1 synthetic DNA or NPM1m genomic DNA (gDNA) extracted from OCI/AML3 cells in a background of wild-type bone marrow (BM) gDNA (range: 0.008%-0.0005% VAF) using a fixed sample input of 400,000 gene copies. DNA sequencing (median read depth: 5.5×106) analysis based on the Inivata MRD platform was performed on the Illumina NextSeq 550 instrument using a proprietary bioinformatic pipeline. A set of follow-up AML samples consisting of peripheral blood (N = 31) and BM (N = 55) from patients enrolled in the UK NCRI AML Study Group AML-19 trial (ISRCTN 2014-002195-90) were tested for comparison of MRD status by NGS with an orthogonal NPM1 RT-qPCR method. This sample cohort included both negative (n = 16) and positive (n = 70) samples as defined by RT-qPCR analysis. To assess the level of agreement between both platforms, we use Pearson's correlation statistics including 95% CIs. Assay performance was evaluated by determining the area under the receiver operating characteristic curve (ROCAUC). RT-qPCR was performed as previously described (Ivey A. N Engl J Med. 2016). NGS results are expressed as percent variant allele frequency (%VAF) and RT-qPCR results as NPM1m transcript copies per 10e4ABL transcripts (normalized copy number [NCN]). Results: The NPM1m NGS assay demonstrated high sensitivity and specificity with a limit of detection with 95% confidence (LoD95) of 0.002% VAF when using serially diluted NPM1 synthetic DNA or NPM1m gDNA extracted from OCI/AML3 cells. In the AML19 set, MRD was detectable in 52/70 (74.3%) samples by both platforms. The level of MRD detected by NGS varied between 0.00025%-46% VAF (median: 0.0042%) and by RT-qPCR between 0.01-1.65x105 NCN (median: 8.03), showing a statistically significant correlation when considering all positive samples (R = 0.84, P < 9.8e-15). Despite this agreement, in 18/70 (25.7%) samples NPM1m was not detected by NGS but detected by RT-qPCR, with NCN between 0.03 and 20.5 (median: 0.37), confirming the expected higher sensitivity of RT-qPCR. Conversely, MRD was detected in 5/16 samples by NGS but not detected by RT-qPCR (VAFs were < technical LoD95 in all cases). The NGS assay could reliably detect MRD positivity in almost all samples with an NCN > 10 copies (23/24 samples, 95.8%) and there was a statistically significant correlation (R = 0.78, P < 1.4e-5). Using a cutoff of > 10 copies, ROC analysis was performed, again demonstrating good concordance (AUC: 91.9%) and identifying an optimal cutoff of 0.003% VAF (specificity = 85.5%, sensitivity = 87.5%). Conclusion: Our DNA-based NGS-MRD assay demonstrates high sensitivity and specificity with a technical LoD95 in serial dilution of 0.002% VAF. It is sufficiently robust to reliably identify MRD positive samples above an RT-qPCR threshold of 10 copies / 10e4ABL (ie, 0.01% NPM1/ABL ratio) and notably, this is tenfold below the provisional European LeukemiaNet definition for high-level MRD of > 0.1%.

Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-β, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

Overexpression of circ_0021739 in Peripheral Blood Mononuclear Cells in Women with Postmenopausal Osteoporosis Is Associated with Reduced Expression of microRNA-194-5p in Osteoclasts.

BackgroundPostmenopausal osteoporosis, a common disease among elderly women, is linked to estrogen deficiency, mechanical loading, and genotype. Circular RNAs (circRNAs) are formed through reverse splicing of the splice donor at the 3′ end and the splice accepter at the 5′ end in pre-mRNA and have been shown to be involved in the development of multiple diseases. Based on their high sequence conservation and stability, circRNAs may be useful biomarkers in different diseases. However, the roles of circRNAs in postmenopausal osteoporosis remain incompletely understood.Material/MethodsFifty-three postmenopausal women were assigned to either the postmenopausal osteoporosis group (n=28) or the control group (n=25). Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the differential expression of circRNAs between the 2 groups. Receiver-operating characteristic (ROC) curve analysis was conducted to evaluate the clinical diagnostic value of circRNA. Prediction of the binding sites between circRNA and miRNAs was conducted using miRanda and RNAhybrid. The function of the circRNA in osteoclastogenesis was determined by circRNA overexpression followed by tartrate-resistant acid phosphatase staining and RT-qPCR analysis.ResultsAmong 4 circRNAs previously identified by RNA-sequencing analysis as differentially expressed in patients with postmenopausal osteoporosis, only hsa_circ_0021739 showed a significant difference in expression between the groups and was downregulated in patients with postmenopausal osteoporosis. The hsa_circ_0021739 expression level was determined to be correlated with the lumbar vertebra, femur, and forearm T-scores. Overexpression of hsa_circ_0021739 decreased the level of hsa-miR-502-5p and inhibited the differentiation of osteoclasts.ConclusionsThe circRNA hsa_circ_0021739 is a potential blood biomarker for postmenopausal osteoporosis. In addition, hsa-miR-502-5p is a likely target of hsa_circ_0021739, which acts to regulate the differentiation of osteoclasts.

Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri.

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiata and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm, ΔCt, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage; PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen); EF1A and ArgK between two populations; β-tubulin and EF1A for different temperatures (5, 15, 25, 35 °C); and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.

Selection of appropriate reference genes for RT-qPCR analysis in Propylea japonica (Coleoptera: Coccinellidae)

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used in molecular biology to analyze RNA expression. The selection of suitable reference genes for data normalization is a precondition for credible measurements of gene expression levels using RT-qPCR. Propylea japonica is one of the most common pests of many crop systems throughout East Asia, and has often been used in the testing of non-target impacts during environmental risk assessments of genetically engineered plants. The present study assessed the suitability of nine frequently used reference genes for comparisons of P. japonica gene expression. Expression stability was compared across developmental stages, sex, a range of tissues, and following exposure to different temperatures. Data were analyzed using RefFinder, which integrated the results obtained using NormFinder, geNorm, BestKeeper, and the ΔCt method. This led to the identification of unique sets of reference genes for each experimental condition: ribosomal protein S18 (RPS18) and elongation factor 1 α (EF1A) for developmental stage comparisons, RPS18 and EF1A for sex comparisons, EF1A and ribosomal protein L4 for tissue comparisons, and RPS18 and EF1A for analyses of temperature-mediated effects. These reference genes will help to enhance the accuracy of RT-qPCR analyses of P. japonica gene expression. This work represents an initial move towards building a standardized system for RT-qPCR analysis of P. japonica, providing a basis for the ecological risk assessment of RNAi-based insect control products.

Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctomaculata.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin, GAPDH, RPL13, RPL6, RPL32, RPS18, and ATPB, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the ΔCt method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata.

Reference gene selection for RT-qPCR analysis in two invasive whiteflies after the acquisition of vectored or non-vectored viruses

Custom reference gene selection is essential for reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in different species of insects and various experiment conditions. In this study, 14 candidate reference genes (HSP40, HSP20, HSP70, HSP90, v-ATPase, RPL29, EF-1, SDHA, Actin, PPIA, GAPDH, MyosinL, NADH, and γ-tubulin) were analyzed using five different programs including ΔCt method, BestKeeper, geNorm, NormFinder, and ReFinder to validate their use as reference genes in two invasive whiteflies, Bemisia tabaci B and Q, after acquiring the vectored virus, Tomato yellow leaf curl virus (TYLCV), or ingesting the non-vectored virus, Tomato spotted wilt virus (TSWV), respectively. The results showed that HSP40, v-ATPase, and EF-1 were the most stable genes in B. tabaci B (B. tabaci B feeding on the healthy, TYLCV- and TSWV-infected tomato plant), PPIA, SDHA, and RPL29 were the most stable genes in B. tabaci Q (B. tabaci Q feeding on the healthy, TYLCV- and TSWV-infected tomato plant). In addition, EF-1, RPL29, and HSP20 were the most stable reference genes in B. tabaci B and Q.These findings provide the basis for future RT-qPCR-based studies on whitefly-virus interactions. Meanwhile, this report may set a precedent for reference gene selection in insects after the ingestion of non-vectored viruses.

BRCA1 expression serves a role in vincristine resistance in colon cancer cells.

The present study aimed to investigate the association between breast cancer susceptibility gene 1 (BRCA1) expression and drug resistance in colon cancer, with the specific aim of elucidating the underlying molecular mechanisms of vincristine (VCR) resistance in tumor cells. The HCT-8 human colon cancer cell line was used to establish the VCR-resistant HCT-8/V line by gradually increasing the concentration of VCR during cell culture. The relative mRNA and protein expression levels of BRCA1 in these colon cancer cell lines was assessed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. Resistance to VCR was established in the HCT-8/V colon cancer cells, and RT-qPCR and western blot analysis revealed the expression of BRCA1 to be significantly higher in the VCR-resistant cells compared with their drug-sensitive counterparts (P<0.05). The decreased BRCA1 expression in these VCR-resistant cells may be associated with the drug resistance frequently observed in colon cancer.

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

Immunohistochemical study and mRNA cytokine profile of the local immune response in cattle naturally infected with Calicophoron daubneyi

In order to recognize the local immune response of the definitive host to Calicophoron daubneyi natural infection, an immunohistochemical study was carried out in the reticulum and rumen in 49 naturally infected cattle. The role of cytokines (IL-4 and IL-10 interleukins and IFN-γ) in the activation of specific defence mechanisms was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays to study cytokine mRNA expression. In all infected animals, CD3+ T lymphocytes seemed to be the main element of the inflammatory infiltrate in the reticular and ruminal lamina propria at the point of the parasite adhesion. Intraepithelial globule leukocytes also showed immunolabelling for CD3. Most CD3+ cells also expressed CD4 (T cell helper) antigen although sporadic CD8+-cytotoxic lymphocytes were observed. Local expression of IFN-γ was observed in damaged papillae at the site of parasite attachment and in scattered cells in the lamina propria. B cells (CD79αcy+, CD45+ and IgG+) were found constantly in relation to lymphoid aggregates. MAC387 was expressed in squamous epithelium and in macrophages of the lamina propria of affected papillae. Macrophages in this location also stained positively for CD163 and CD68. Intraepithelial Langerhans cells and macrophages located in the lamina propria showed immunopositivity for MHCII in the affected areas. RT-qPCR analysis confirmed a statistical significant increase of IFN-γ, and IL-10 expression (p<0.01) in the rumen associated with the presence of flukes. These findings suggest a predominant Th1 polarized local immune response with the probable involvement of Th regulatory cells in cattle C. daubneyi natural infection.

Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

A Comprehensive Selection of Reference Genes for RT-qPCR Analysis in a Predatory Lady Beetle, Hippodamia convergens (Coleoptera: Coccinellidae).

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable, rapid, and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate RT-qPCR data, normalization relative to stable reference genes is required. In this study, expression profiles of seven candidate reference genes, including β-actin (Actin), elongation factor 1 α (EF1A), glyceralde hyde-3-phosphate dehydro-genase (GAPDH), cyclophilins A (CypA), vacuolar-type H+-ATPase (ATPase), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S) from Hippodamia convergens were investigated. H. convergens is an abundant predatory species in the New World, and has been widely used as a biological control agent against sap-sucking insect pests, primarily aphids. A total of four analytical methods, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to evaluate the performance of these seven genes as endogenous controls under diverse experimental conditions. Additionally, RefFinder, a comprehensive evaluation platform integrating the four above mentioned algorithms, ranked the overall stability of these candidate genes. A suite of reference genes were specifically recommended for each experimental condition. Among them, 28S, EF1A, and CypA were the best reference genes across different development stages; GAPDH, 28S, and CypA were most stable in different tissues. GAPDH and CypA were most stable in female and male adults and photoperiod conditions, 28S and EF1A were most stable under a range of temperatures, Actin and CypA were most stable under dietary RNAi condition. This work establishes a standardized RT-qPCR analysis in H. convergens. Additionally, this study lays a foundation for functional genomics research in H. convergens and sheds light on the ecological risk assessment of RNAi-based biopesticides on this non-target biological control agent.

Evaluation of Reference Genes for RT-qPCR in Tribolium castaneum (Coleoptera: Tenebrionidae) Under UVB Stress.

Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) has become a widely used technique to quantify gene expression. It is necessary to select appropriate reference genes for normalization. In the present study, we assessed the expression stability of seven candidate genes in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) irradiated by ultraviolet B (UVB) at different developmental stages for various irradiation time periods. The algorithms of geNorm, NormFinder, and BestKeeper were applied to determine the stability of these candidate genes. Ribosomal protein genes RpS3, RpL13A, and β-actin gene (ActB) showed the highest stability across all UVB irradiation time points, whereas expression of other normally used reference genes, such as those encoding the β-tubulin gene TUBB and the E-cadherin gene CAD, varied at different developmental stages. This study will potentially provide more suitable reference gene candidates for RT-qPCR analysis in T. castaneum subjected to environmental stresses, particularly UV irradiation.