Bamboo was one of the first plants to be cultivated in China and is widely used in industry and daily life. The study of gene function has become an important part of bamboo breeding, whereas ...quantitative real-time PCR (qRT-PCR) is a powerful tool for gene expression analysis. The accuracy of qRT-PCR results largely depends on suitable reference genes. In this study, a transcriptome-wide identification of reference genes was conducted based on 447 transcriptome datasets, comprising 200 tissue samples, 107 treated samples, and 140 samples from various moso bamboo (Phyllostachys edulis) forms. A total of 3444, 1013, and 3962 stably expressed genes were identified from these three groups, respectively. Functional enrichment analysis revealed significant enrichment of these genes in pathways, including the spliceosome, proteasome, and oxidative phosphorylation. Eight candidate genes (ADPRE, GAPDH, TRX, TUBA, NRP, MBF, UNK, and CAM1), were selected for qRT-PCR validation using 112 samples. To assess their stability, five statistical methods (geNorm, NormFinder, BestKeeper, Delta-Ct, and RefFinder) were employed. The most suitable reference genes were ADPRE and GAPDH for different tissues, GAPDH and CAM1 for different treatments, and GAPDH and TRX for various moso bamboo forms. Overall, ADPRE and GAPDH were the most stable reference genes across all conditions, while TUBA and TRX were the least stable reference genes. In addition, a significant negative correlation was found between the Ct values of RT-qPCR and the log2TPM values from the transcriptome data (Ct = −1.534x + 37.221), providing a potential method for estimating gene expression levels. The identified reference genes, particularly ADPRE and GAPDH, provide a robust set of references for gene expression studies in moso bamboo.
Flavanone 3-hydroxylase (F3H) plays a crucial role in the biosynthesis of flavonoids. In the present study, one F3H gene (P_edulia040010337.g) from Passiflora edulis Sims, which has a coding sequence ...(CDS) of 1161 bp, encoding a protein consisting of 386 amino acid residues was cloned. The PeF3H protein contains a non-heme dioxygenase (DIOX-N superfamily) domain and a typical F3H protein functional domain (2OG-FeII-Oxy dioxygenase). Phylogenetic analysis revealed that the PeF3H protein shared high similarity with F3H proteins in Turnera subulata, Populus alba, and Populus tomentosa, with 88% identities of amino acid sequences. The PeF3H protein lacks a transmembrane structure, indicating it is likely to be expressed in the mitochondria. Additionally, 3D structure, protein and protein interaction, and KEGG pathway of PeF3H were anticipated based on homologous proteins. qRT-PCR analysis showed that PeF3H was highly expressed in leaves, followed by stems and roots. These studies have provided insights into the molecular mechanisms underlying flavonoid biosynthesis and predicted potential targets for genetic engineering to improve the nutritional and medicinal properties of passion fruit.
Bangladesh J. Bot. 52(2): 613-623, 2023 (June) Special
Human norovirus (HuNoV) is a leading cause of foodborne gastroenteritis. Unfortunately, the inactivation parameters for HuNoV in clinical, food and environmental samples have not been established. ...Due to the inability to cultivate HuNoV in vitro, quantitative real-time RT-PCR (qRT-PCR) is widely-used for detecting HuNoVs. However, qRT-PCR does not indicate viral infectivity. Our method employs histo-blood group antigens (HBGAs) as viral receptors/co-receptors and container-affixed capture agents to concentrate HuNoVs. The captured viruses are denatured and its genome is amplified in the same module by in situ capture qRT-PCR (ISC-qRT-PCR). Greater than three log10 reduction in the receptor-captured viral genomic signal (RCVGS) was observed when HuNoV was treated by heat at 72°C for 4min, by chlorine at a final concentration of 16ppm in less than 1min, and by UV irradiation at 1J/cm2. Treatment of low-titer HuNoV (<103 copies/sample) with 70% ethanol for 20s reduced the RCVGS of HuNoV by two log10. However, ethanol had a limited effect on high-titer samples of HuNoV (>103 copies/sample). The results demonstrate that ISC-qRT-PCR method could be used as an alternative method to measure encapsidated viral RNA and indirectly indicate the inactivation status of HuNoV caused by physical treatment such as heat, and chemical treatment such as chlorine, that damage the ability of the virus to bind to its receptor.
•The inactivation parameters for HuNoV have not been established.•An in situ capture qRT-PCR method was developed to determine inactivation of HuNoV.•ISC-qRT-PCR provides an alternative method to measure infectivity of HuNoV.•HuNoV inactivation parameters were: 72°C (4min), 16ppm chlorine or 1J/cm2 UV.•EtOH had a limited effect on HuNoV.
Soil salinity is the major abiotic stress that disrupts nutrient uptake, hinders plant growth, and threatens agricultural production. Plant growth-promoting rhizobacteria (PGPR) are the most ...promising eco-friendly beneficial microorganisms that can be used to improve plant responses against biotic and abiotic stresses. In this study, a previously identified
B. thuringiensis
PM25 showed tolerance to salinity stress up to 3 M NaCl. The Halo-tolerant
Bacillus thuringiensis
PM25 demonstrated distinct salinity tolerance and enhance plant growth-promoting activities under salinity stress. Antibiotic-resistant Iturin C (
ItuC
) and bio-surfactant-producing (
sfp
and
srfAA
) genes that confer biotic and abiotic stresses were also amplified in
B. thuringiensis
PM25. Under salinity stress, the physiological and molecular processes were followed by the over-expression of stress-related genes (APX and SOD) in
B. thuringiensis
PM25. The results detected that
B. thuringiensis
PM25 inoculation substantially improved phenotypic traits, chlorophyll content, radical scavenging capability, and relative water content under salinity stress. Under salinity stress, the inoculation of
B. thuringiensis
PM25 significantly increased antioxidant enzyme levels in inoculated maize as compared to uninoculated plants. In addition,
B. thuringiensis
PM25-inoculation dramatically increased soluble sugars, proteins, total phenols, and flavonoids in maize as compared to uninoculated plants. The inoculation of
B. thuringiensis
PM25 significantly reduced oxidative burst in inoculated maize under salinity stress, compared to uninoculated plants. Furthermore,
B. thuringiensis
PM25-inoculated plants had higher levels of compatible solutes than uninoculated controls. The current results demonstrated that
B. thuringiensis
PM25 plays an important role in reducing salinity stress by influencing antioxidant defense systems and abiotic stress-related genes. These findings also suggest that multi-stress tolerant
B. thuringiensis
PM25 could enhance plant growth by mitigating salt stress, which might be used as an innovative tool for enhancing plant yield and productivity.
Information on the impact of hormonal protocols for cervical dilation on the quality of ovine embryos is scarce.
To compare the quality of embryos after cervical dilation protocol, ewes (n = 64) were ...allocated into either a treated group (100 μg estradiol benzoate intravenous and 0.12 mg cloprostenol intramuscularly, 12 hours before embryo collection plus 100 iu oxytocin intravenous 15 minutes before the collection procedure) or a control group (saline). Luteal function was analysed using ultrasonography and P4 measurement. Some collected embryos were frozen/thawed for gene expression, others were cultured in vitro, frozen/thawed for gene expression, and the remaining embryos were fixed for the apoptosis test (TUNEL test).
The treatment reduced fluid (p=0.04) and structure (p=0.03) recovery rates, but the morphological quality, development stage, and apoptosis incidence of the embryos were not affected by treatment. The corpora lutea of the control group had greater blood perfusion (p = 0.002) and greater P4 concentrations at 6, 9, and 12 h after the treatment (p < 0.0001). The expression of BAX, BCL2, PRDX1, and HSP90 genes were not affected by the treatment. However, the embryos in the treated group had fewer NANOG and OCT4 transcripts than control embryos (p = 0.008; p = 0.006, respectively). After culture, there was no difference between the groups in any gene.
The hormonal protocol for cervical dilation reduced the efficiency of embryo collection. In addition, the treatment induced luteolysis and a transient alteration of embryo gene expression, however there were no detectable changes in embryo morphological quality, development stage, or incidence of apoptosis.
Alternative splicing is a complex process that contributes to the generation of diverse mRNA and protein isoforms, including in oil palm (Elaeis guineensis). Despite their importance, many functions ...of alternative splicing genes remain poorly characterized. This study aims to investigate splicing variants of gene encoding Heading date 3a in E. guineensis (EgHd3a) using the GenBank database and ClustalW algorithm. To ensure the data accuracy and reliability of design isoform‐ specific primers, special emphasis is given to primer design techniques and validation using polymerase chain reaction (PCR) and quantitative real‐time (qRT)‐PCR analysis. The designed primers demonstrated high specificity and discrimination between mRNA specimens. Nucleotide variations at the 3’‐end influenced the specificity of primers with the addition of GC composition. Furthermore, qRT‐PCR analysis revealed a strong correlation between Ct values and gene concentration for the isoforms which indicates a reliable amplification of EgHd3a. Although two isoforms, Hd3a‐X2 and Hd3a‐X3, showed slightly higher than acceptable PCR efficiency values, caution is advised to prevent non‐specific amplification. Despite the challenge posed by the limitation of primer positioning due to alternative splicing, the chosen primer proved optimal for analysis. This study highlights the importance of considering alternative splicing in gene quantification experiments and provides insights into the critical steps, methods, and quality control measures necessary for accurately detecting alternative splicing events, contributing to understanding this complex biological process.
The development of portable systems for analysis of nucleic acids (NAs) is crucial for the evolution of biosensing in the context of future healthcare technologies. The integration of NA extraction, ...purification, and detection modules, properly actuated by microfluidics technologies, is a key point for the development of portable diagnostic systems. In this paper, we describe an integrated biosensor platform based on a silicon–plastic hybrid lab‐on‐disk technology capable of managing NA extraction, purification, and detection processes in an integrated format. The sample preparation process is performed by solid‐phase extraction technology using magnetic beads on a plastic disk, while detection is done through quantitative real‐time polymerase chain reaction (qRT‐PCR) on a miniaturized silicon device. The movement of sample and reagents is actuated by a centrifugal force induced by a disk actuator instrument. The assessment of the NA extraction and detection performance has been carried out by using hepatitis B virus (HBV) DNA genome as a biological target. The quantification of the qRT‐PCR chip in the hybrid disk showed an improvement in sensitivity with respect to the qRT‐PCR commercial platforms, which means an optimization of time and cost. Limit of detection and limit of quantification values of about 8 cps/reaction and 26 cps/reaction, respectively, were found by using analytical samples (synthetic clone), while the results with real samples (serum with spiked HBV genome) indicate that the system performs as well as the standard methods.
The integration on nucleic acids extraction, purification and detection modules is a key point for the development of portable system for molecular diagnostics. Here we described an integrated biosensor platform based on hybrid Lab‐on‐Disk for the qRT‐PCR detection of pathogen nucleic acids.
Plants possess a well-organized protective network, wherein antioxidant enzymes play an important part in dealing with oxidative stress induced by over accumulation of ROS in plant cells. In the ...present study, a microcosm hydroponic experiment was performed to investigate the molecular modification of antioxidant enzymes at subcellular levels in rice seedlings in the presence of either trivalent Cr(III) or hexavalent chromium Cr(VI) using rice oligonucleotide microarray analysis. The results indicated that the production of ROS induced by Cr(III, VI) was concentration-dependent, Cr-specific and tissue-specific. Trivalent or hexavalent chromium exposure significantly (p < 0.05) altered the antioxidant enzymes activities in both rice tissues in comparison to control plants. In total, 41 genes were identified from the data of rice oligonucleotide microarray analysis. Under Cr(III) exposure, relatively higher expression of genes was observed in roots compared to those in shoots (p < 0.05), while gene expressions in both plant parts differed slightly during Cr(VI) exposure, implying different regulation and response strategies of plants against Cr(III) and Cr(VI). Subcellular localization indicated that genes encoding SOD, POD, APX, and GPX are mainly prevalent in the cytoplasm (30.77%), chloroplasts (29.23%), peroxisomes (10.77%) and mitochondria (9.23%), suggesting that cytoplasm and chloroplasts are the main sites responsible for scavenging ROS through enzymatic processes. Our study provides new insight into the roles of antioxidant enzymes in ROS metabolism at subcellular levels under Cr exposure.
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•Generation of ROS induced by Cr was concentration-dependent and tissue-specific.•Cr exposure altered the activities of antioxidant enzymes in both roots and shoots of rice plants.•41 antioxidant enzymatic genes corresponding to Cr were identified by microarray analysis.•A gene regulation diagram of eight antioxidant enzymes at the subcellular levels is proposed.•Genes encoding antioxidant enzymes are predominant in the cytoplasm and chloroplast.
Pancreatic cancer (PC) has posed a great health threat to a growing number of people all over the world. Detection of serum miRNAs, being sensitive, noninvasive, and easy to obtain, has a great ...potential of being a novel screening method for PC patients. In this study, we investigated miRNA expression levels in serum by qRT‐PCR. The study was divided into four phases: the screening, training, testing, and external validation stage. We firstly chose candidate miRNAs using Exiqon panels in the screening phase. Then, a total of 129 PC serum samples and 107 normal controls (NCs) were further analyzed in the following training and testing phases to identify differently expressed miRNAs. A cohort of 30 PC serum samples vs 30 NCs was used to confirm the diagnostic value of the identified miRNAs in the external validation phase. Moreover, miRNA expressions in additional 44 PC tumor tissue samples and the matched adjacent normal tissue samples as well as 32 pairs of serum‐derived exosomes samples were also further explored. As a result, we identified six significantly upregulated miRNAs in the serum of PC: let‐7b‐5p, miR‐192‐5p, miR‐19a‐3p, miR‐19b‐3p, miR‐223‐3p, and miR‐25‐3p. A six‐miRNA panel in serum was then established. The area under the receiver operating characteristic curves (AUC) for the panel was 0.910 for the combined training and testing phases, which showed higher diagnostic value than the individual miRNA. Prognostic value prediction using Cox's proportional hazards model and Kaplan‐Meier curves showed that increased serum miR‐19a‐3p was closely related to worse overall survival (OS). In addition, significant upregulation of miR‐192‐5p, miR‐19a‐3p, and miR‐19b‐3p was observed in both PC tissue and serum‐derived exosomes samples. In conclusion, we identified a six‐miRNA (let‐7b‐5p, miR‐192‐5p, miR‐19a‐3p, miR‐19b‐3p, miR‐223‐3p, and miR‐25‐3p) panel in the serum for PC early and noninvasive diagnosis.
We identified a six‐miRNA (let‐7b‐5p, miR‐192‐5p, miR‐19a‐3p, miR‐19b‐3p, miR‐223‐3p, and miR‐25‐3p) panel in the serum by multiple‐phase validation using qRT‐PCR for PC early and noninvasive diagnosis.
Circulating microRNAs are promising biomarkers for non‐invasive testing and dynamic monitoring in cancer patients. However, no consensus exists regarding the normalization of circulating microRNAs in ...the quantification, making the results incomparable. We investigated global circulating microRNA profiles to identify a stable endogenous control for quantifying circulating microRNAs using three cohorts (n = 544), including 168 control individuals (healthy subjects and those with chronic hepatitis B and cirrhosis) and 376 cancer patients (hepatocellular, colorectal, lung, esophageal, gastric, renal, prostate, and breast cancer patients). GeNorm, NormFinder, and coefficient of variability (CV) were used to select the most stable endogenous control, whereas Ingenuity Pathway Analysis (IPA) was adopted to explore its signaling pathways. Seven candidates (miR‐1225‐3p, miR‐1228, miR‐30d, miR‐939, miR‐940, miR‐188‐5p, and miR‐134) from microarray analysis and four commonly used controls (miR‐16, miR‐223, let‐7a, and RNU6B) from literature were subjected to real‐time quantitative reverse transcription‐polymerase chain reaction validation using independent cohorts. MiR‐1228 (CV = 5.4%) with minimum M value and S value presented as the most stable endogenous control across eight cancer types and three controls. IPA showed miR‐1228 to be involved extensively in metabolism‐related signal pathways and organ morphology, implying that miR‐1228 functions as a housekeeping gene. Functional network analysis found that “hematological system development” was on the list of the top networks that associate with miR‐1228, implying that miR‐1228 plays an important role in the hematological system. The results explained the steady expression of miR‐1228 in the blood. In conclusion, miR‐1228 is a promising stable endogenous control for quantifying circulating microRNAs in cancer patients.
What's new?
While circulating microRNAs (miRNAs) are promising cancer biomarkers, a standard control for the normalization of serum/plasma miRNA levels is yet to be established. Without such a control, data from different studies and different cancers remains incomparable. Here, analysis of global circulating microRNA profiles in healthy individuals and cancer patients suggests that miR‐1228, one of seven candidates identified from microarray analysis, is a stable endogenous control for the quantification of circulating miRNAs in cancer patients. MiR‐1228 allows for the comparison of circulating miRNA expressions in the same cancer across different studies and in different cancers of the same study.
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