scholarly journals Reconstruction of insect hormone pathways in an aquatic firefly, Sclerotia aquatilis (Coleoptera: Lampyridae), using RNA-seq

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7428 ◽  
Author(s):  
Pornchanan Chanchay ◽  
Wanwipa Vongsangnak ◽  
Anchana Thancharoen ◽  
Ajaraporn Sriboonlert
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Biological Databases ◽  
Rt Pcr ◽  
Kegg Pathways ◽  
Insect Hormones ◽  
Insect Metamorphosis

Insect hormones: ecdysteroids and juvenile hormones have crucial functions during the regulation of different developmental pathways in insects. Insect metamorphosis is one of the primary pathways regulated by these hormones. The insect hormone biosynthetic pathway is conserved among arthropods, including insects, with some variations in the form of hormones used among each group of insects. In this study, the candidate genes involved in the insect hormone pathways and their functional roles were assessed in an aquatic firefly, Sclerotia aquatilis using a high-throughput RNA sequencing technique. Illumina next-generation sequencing (NGS) was used to generate transcriptome data for the different developmental stages (i.e., larva, pupa, and adult) of S. aquatilis. A total of 82,022 unigenes were generated across all different developmental stages. Functional annotation was performed for each gene, based on multiple biological databases, generating 46,230 unigenes. These unigenes were subsequently mapped using KEGG pathways. Accordingly, 221 protein-encoding genes involved in the insect hormone pathways were identified, including, JHAMT, CYP15A1, JHE, and Halloween family genes. Twenty potential gene candidates associated with the biosynthetic and degradation pathways for insect hormones were subjected to real-time PCR, reverse transcriptase PCR (RT-PCR) and sequencing analyses. The real-time PCR results showed similar expression patterns as those observed for transcriptome expression profiles for most of the examined genes. RT-PCR and Sanger sequencing confirmed the expressed coding sequences of these gene candidates. This study is the first to examine firefly insect hormone pathways, facilitating a better understanding of firefly growth and development.

103 GENE EXPRESSION PROFILES OF VITRIFIED MOUSE EMBRYOS DETECTED BY MICROARRAYS

2006 ◽  
Vol 18 (2) ◽  
pp. 160
Author(s):  
S. Mamo ◽  
Sz. Bodo ◽  
Z. Polgar ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Differentially Expressed ◽  
Analysis Tool ◽  
Cell Stage ◽  
Base Medium ◽  
Independent Analysis

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to examine the transcript variations and identify genes most affected by the treatment. For this, 8-cell-stage embryos were collected from female ICR mice mated with ICR males. The embryos were washed with CZB-HEPES base medium and suspended briefly in equilibrium medium consisting of 4% ethylene glycol (EG) in base medium at room temperature. Following equilibration, the embryos were vitrified in a 35% EG, 0.4 M trehalose, 5% polyvinylpyrrolidone (PVP) solution by means of a solid-surface vitrification (SSV) technique as described earlier (Dinnyes 2000 Biol. Reprod. 63, 513-518). Then 40 embryos each from the control and the vitrified/warmed groups were cultured in CZB medium for 3 h. Total RNAs were extracted from cultured embryos in each group using TRIzol (Invitrogen, Bio-Science, Ltd., Budapest, Hungary), following the manufacturer's instructions. Two rounds of amplification were employed to produce labeled RNA, using low input RNA amplification kit (Agilent Technologies, Kromat, Ltd., Budapest, Hungary) procedures with modifications. Three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22K oligonucleotide slides with subsequent analysis of the results. Moreover, as an independent analysis tool, real time PCR was used with eight designed primers. All of the vitrified embryos were recovered after warming with no morphological signs of cryodamage and used for analysis. The two rounds of amplification yielded 15-16 �g of cRNA. The analysis of repeated hybridizations by Rosetta luminator software (Agilent) showed 20 183 genes and expressed sequence tags (ESTs) that passed the selection criteria and were identified as common signatures in all of the slides. Unsupervised analysis of the gene expression data identified a total of 631 differentially expressed (P < 0.01) genes. However, to support the reliability of the results, only those variations above 1.5 fold differences were considered as significant in the final analysis. Therefore, with this stringent criterion 183 genes were differentially expressed (P < 0.01), of which 109 were up-regulated and the remainder down-regulated. Although genes have multiple and overlapping functions, most of the differentially expressed genes were functionally classified into various physiological categories. These include stress response (8), apoptosis related (6), metabolism (51), temperature response (4), and transcription regulation (15). Moreover, the independent analysis with real time PCR and unamplified samples verified the results of microarray. Thus, based on confirmation of the results by an independent analysis and support by the previous studies for some of the genes, it is possible to conclude that the expression patterns reflect the true biological image of embryos after vitrification, with most effects on stress- and cell metabolism-related genes. This work was supported by EU FP6 (MEXT-CT-2003-59582), Wellcome Trust Foundation (Grant No. 070246), and National Office of Research and Technology (NKTH) (#BIO-00017/2002, #BIO-00086/2002).

scholarly journals Selection and Validation of Reference Genes for Quantitative Real-Time PCR Expression Analysis of Candidate Genes in Carposina sasakii (Lepidoptera: Carposinidae)

2020 ◽  
pp. 75-85
Author(s):  
Zuobing Yan ◽  
Yongli Li ◽  
Zhou Zhou ◽  
Yongan Zhang ◽  
Liangjian Qu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Quantitative Real Time Pcr ◽  
Carposina Sasakii

Carposina sasakii is one of the most important pests on the quality of stone and pome fruits. Investigation of a gene expression level in the species is hampered because of the gap of validated reference genes. The expression variation in the transcription levels of eight candidate reference genes, Actin (ACT), Tubulinbeta-1 (TUB), Ribosomal protein 49 (RP49), Elongation factor1-alpha (EF-1a), Elongation factor1-b (EF-1b), Elongation factor1-d (EF-1d), Ribosomal proteinL13 (RPL13) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were analyzed by quantitative real-time PCR (qPCR). The stability and ranking of these gene expression profiles in three organ types (head, thorax and abdomen), three developmental stages (larva, pupa and moth), and five diapause states (non-diapause, pre-diapause, diapause 0 d, diapause 20 d and diapause 60 d) were assessed using two algorithm-based methods, geNorm and NormFinder. EF-1a, ACT and GAPDH were evaluated to be the three stable reference genes based on the important observations and comprehensive analysis, whereas TUB and EF-1b showed low expression stability. Best gene combinations for different qPCR analysis in C. sasakii could be chosen from the three stable reference genes, the using of two reference genes is sufficient to effectively normalize qPCR data in C. sasakii. The study laid the foundation for gene expression analysis in C. sasakii and provided new information for the selection of reference genes.

scholarly journals Temporal and Spatial Expression Patterns of Bone Morphogenetic Protein 3 in Developing Zebrafish

2014 ◽  
Vol 8 (1) ◽  
pp. 69-72 ◽  
Author(s):  
Midori Ito-Amano ◽  
Yukio Nakamura ◽  
Mika Morisaki ◽  
Xinjun He ◽  
Masanori Hayashi ◽  
...  
Keyword(s):  
Real Time ◽  
Bone Morphogenetic Proteins ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Otic Vesicle ◽  
Bone Biology ◽  
Pharyngeal Arches ◽  
Morphogenetic Protein

Bone morphogenetic proteins (BMPs) are important elements in bone biology. We herein report the expression profiles of zebrafish bmp3 (zbmp3) as demonstrated by real-time PCR and in situ hybridization. The expression of zbmp3 was highly detectable by real-time PCR from 1 day post-fertilization (1 dpf) to 2 weeks post-fertilization (2 wpf) and peaked at 1 wpf. For in situ hybridization experiments, zbmp3 was expressed in the otic vesicle at 1 dpf, 2 dpf, 3 dpf, and 5 dpf. It was also expressed in the pharyngeal arches, including the opercle, branchiostegal ray, and pectoral fins, at 2 dpf. Our results suggest that zbmp3 may play an important role in the skeletal biology of developing zebrafish.

scholarly journals Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

2022 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohamed Hazman
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Express Method ◽  
Relative Expression ◽  
Crossing Point

Abstract Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

scholarly journals Genome-Wide Analysis and Characterization of the Aux/IAA Family Genes Related to Floral Scent Formation in Hedychium coronarium

2019 ◽  
Vol 20 (13) ◽  
pp. 3235 ◽  
Author(s):  
Yanguo Ke ◽  
Farhat Abbas ◽  
Yiwei Zhou ◽  
Rangcai Yu ◽  
Yuechong Yue ◽  
...  
Keyword(s):  
Gene Family ◽  
Developmental Stages ◽  
Floral Scent ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Virus Induced Gene Silencing ◽  
Cut Flowers ◽  
Flower Opening ◽  
Hedychium Coronarium

Auxin plays a key role in different plant growth and development processes, including flower opening and development. The perception and signaling of auxin depend on the cooperative action of various components, among which auxin/indole-3-acetic acid (Aux/IAA) proteins play an imperative role. In a recent study, the entire Aux/IAA gene family was identified and comprehensively analyzed in Hedychium coronarium, a scented species used as an ornamental plant for cut flowers. Phylogenetic analysis showed that the Aux/IAA gene family in H. coronarium is slightly contracted compared to Arabidopsis, with low levels of non-canonical proteins. Sequence analysis of promoters showed numerous cis-regulatory elements related to various phytohormones. HcIAA genes showed distinct expression patterns in different tissues and flower developmental stages, and some HcIAA genes showed significant responses to auxin and ethylene, indicating that Aux/IAAs may play an important role in linking hormone signaling pathways. Based on the expression profiles, HcIAA2, HcIAA4, HcIAA6 and HcIAA12, were selected as candidate genes and HcIAA2 and HcIAA4 were screened for further characterization. Downregulation of HcIAA2 and HcIAA4 by virus-induced gene silencing in H. coronarium flowers modified the total volatile compound content, suggesting that HcIAA2 and HcIAA4 play important roles in H. coronarium floral scent formation. The results presented here will provide insights into the putative roles of HcIAA genes and will assist the elucidation of their precise roles during floral scent formation.

scholarly journals Transcriptional Profiles of Secondary Metabolite Biosynthesis Genes and Cytochromes in the Leaves of Four Papaver Species

Data ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. 55 ◽  
Author(s):  
Dowan Kim ◽  
Myunghee Jung ◽  
In Ha ◽  
Min Lee ◽  
Seok-Geun Lee ◽  
...  
Keyword(s):  
Secondary Metabolite ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Bioinformatics Analyses ◽  
Kegg Pathways ◽  
Benzylisoquinoline Alkaloid ◽  
Development Life Cycle ◽  
The Family ◽  
Leaf Transcriptome ◽  
Secondary Metabolite Biosynthesis

Poppies are well-known plants in the family Papaveraceae that are rich in alkaloids. This family contains 61 species, and in this study we sequenced the transcriptomes of four species’ (Papaver rhoeas, Papaver nudicaule, Papaver fauriei, and Papaver somniferum) leaves. These transcripts were systematically assessed for the expression of secondary metabolite biosynthesis (SMB) genes and cytochromes, and their expression profiles were assessed for use in bioinformatics analyses. This study contributed 265 Gb (13 libraries with three biological replicates) of leaf transcriptome data from three Papaver plant developmental stages. Sequenced transcripts were assembled into 815 Mb of contigs, including 226 Mb of full-length transcripts. The transcripts for 53 KEGG pathways, 55 cytochrome superfamilies, and benzylisoquinoline alkaloid biosynthesis (BIA) were identified and compared to four other alkaloid-rich genomes. Additionally, 22 different alkaloids and their relative expression profiles in three developmental stages of Papaver species were assessed by targeted metabolomics using LC-QTOF-MS/MS. Collectively, the results are given in co-occurrence heat-maps to help researchers obtain an overview of the transcripts and their differential expression in the Papaver development life cycle, particularly in leaves. Moreover, this dataset will be a valuable resource to derive hypotheses to mitigate an array of Papaver developmental and secondary metabolite biosynthesis issues in the future.

Genome-wide screening of m6A-modified transcript in the Wilms’ tumor

2021 ◽  
Author(s):  
Zhuo Liu ◽  
Feng He ◽  
Jing Liu ◽  
Shengrong OuYang ◽  
Zexi Li ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Real Time ◽  
Real Time Pcr ◽  
Wilms Tumor ◽  
Expression Patterns ◽  
Mrna Levels ◽  
Gene Ontology Analysis ◽  
Quantitative Real Time Pcr ◽  
Related Factors ◽  
Significant Difference

Abstract Background Wilms’ tumor, also called nephroblastoma, is the most common pediatric renal malignancy. The pathogenesis of Wilms’ tumor has been attributed to several genetic and epigenetic factors. However, the most pervasive internal mRNA modification that affects almost every process of RNA metabolism, RNA N6-Methyladenosine (m6A) methylation, has not been characterized in Wilms’ tumor. Methods Wilms’ tumor (WT) and adjacent non-cancerous (NC) tissue samples were obtained from 23 children with nephroblastoma, and the global m6A levels were measured by mass spectrometry. Analyses by m6A-mRNA epitranscriptomic microarray and mRNA microarray were performed, and m6A-related mRNAs were validated by quantitative real-time PCR for input and m6A-immunoprecipitated RNA samples from WT and NC tissues. Gene ontology analysis and KEGG pathway analysis were performed for differentially expressed genes, and expression of RNA methylation-related factors was measured by quantitative real-time PCR. Results The total m6A methylation levels in total RNA of WT samples and NC samples were (0.21 ± 0.01)% and (0.22 ± 0.01)%, respectively, with no statistically significant difference. Fifty-nine transcripts were differentially m6A-methylated between the WT and NC groups, which showed distinct m6A modification patterns. Gene ontology analysis indicated that m6A-modified genes were enriched in cancer-associated pathways, including the mTOR pathway, and conjoint analysis of the unique methylation and gene expression patterns in WT samples suggested an association with metabolic pathways.The mRNA levels of the m6A-related “reader” genes, YTHDF1, YTHDF2 and IGF2BP3, were statistically higher in WT samples than in NC samples. Conclusion This is the first study to determine the m6A modification profiles in Wilms’ tumor. Our data provide novel information regarding patterns of m6A modification that correlate with carcinogenesis in Wilms’ tumor.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

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