Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing

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.

Download Full-text

Cloning, Characterization, and Expression Features of Chicken CDS2 Splicing Variants

10.21203/rs.3.rs-80587/v1 ◽

2020 ◽

Author(s):

Yuanyuan Xu ◽

Shuping Zhang ◽

Yujun Guo ◽

Wen Chen ◽

Yanqun Huang

Keyword(s):

Adipose Tissue ◽

Real Time ◽

Real Time Pcr ◽

Expression Patterns ◽

Mrna Levels ◽

Exogenous Insulin ◽

Quantitative Real Time Pcr ◽

Temporal Expression ◽

Spatio Temporal ◽

The Brain

Abstract Background: The CDS gene encodes the CDP-diacylglycerol synthase enzyme that catalyzes the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid. At present, there are no reports of CDS2 in birds. Here, we identified chicken CDS2 transcripts by combining conventional RT- PCR amplification, 5' RACE (Fig. 1A), and 3' RACE, explored the spatio-temporal expression profiles of total CDS2 and the longest transcript variant CDS2-4, and investigated the effect of exogenous insulin on total the mRNA level of CDS2 by quantitative real-time PCR. Results: Four transcripts of chicken CDS2 (CDS2-1, -2, -3, and -4) were identified, which were alternatively spliced at the 3′-untranslated region (UTR). CDS2 was widely expressed in all tissues examined and the longest variant CDS2-4 was the major transcript. Both total CDS2 and CDS2-4 were prominently expressed in adipose tissue and the heart, and exhibited low expression in the liver and pectoralis of 49 day-old chickens. Quantitative real-time PCR revealed that total CDS2 and CDS2-4 had different spatio-temporal expression patterns in chicken. Total CDS2 exhibited a similar temporal expression tendency with a high level in the later period of incubation (embryonic day 19 [E19] or 1-day-old) in the brain, liver, and pectoralis. While CDS2-4 presented a distinct temporal expression pattern in these tissues, CDS2-4 levels peaked at 21 days in the brain and pectoralis, while liver CDS2-4 mRNA levels were highest at the early stage of hatching (E10). Total CDS2 (P < 0.001) and CDS2-4 (P = 0.0090) mRNA levels in the liver were differentially regulated throughout development of the chicken. Exogenous insulin significantly downregulated the level of total CDS2 at 240 min in the pectoralis of Silky chickens (P < 0.01). Total CDS2 levels in the liver of Silky chickens were higher than that of the broiler in the basal state and after insulin stimulation. Conclusion: Chicken CDS2 has multiple transcripts with variation at the 3′-UTR, which was prominently expressed in adipose tissue. Total CDS2 and CDS2-4 presented distinct spatio-temporal expression patterns, and they were differentially regulated with age in liver. Insulin could regulate chicken CDS2 levels in a breed- and tissue-specific manner.

Download Full-text

Circular RNA sequencing indicates circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers of primary Sjögren’s syndrome

Rheumatology ◽

10.1093/rheumatology/keaa163 ◽

2020 ◽

Vol 59 (9) ◽

pp. 2603-2615 ◽

Cited By ~ 2

Author(s):

Fengxia Li ◽

Zhenwei Liu ◽

Bing Zhang ◽

Shan Jiang ◽

Qiongdan Wang ◽

...

Keyword(s):

Receiver Operating Characteristic ◽

Real Time ◽

Rna Sequencing ◽

Clinical Features ◽

Real Time Pcr ◽

Operating Characteristic ◽

Receiver Operating Characteristic Analysis ◽

Quantitative Real Time Pcr ◽

Characteristic Analysis ◽

Noninvasive Biomarkers

Abstract Objectives This study aims to characterize the expression profiles of circRNAs in primary Sjogren’s Syndrome (pSS) and examine the potential of noninvasive circular RNAs (circRNAs) as biomarkers of pSS. Methods We performed RNA sequencing of minor salivary gland (MSG) biopsies from four pSS and four non-pSS individuals (subjects undergoing MSG biopsies but not meeting 2012 or 2016 ACR classification criteria for SS). Differentially expressed circRNAs were identified by DESeq2, and confirmed by quantitative real-time PCR in the MSGs as well as in plasma exosomes in 37 pSS and 14 non-pSS subjects. Discriminatory capacity testing using receiver operating characteristic analysis was used to evaluate the performance of circRNAs as diagnostic biomarkers for pSS. Results Circ-IQGAP2 and circ-ZC3H6 had significantly upregulated expression in the MSGs of pSS patients, and this elevated expression was confirmed by quantitative real-time PCR of plasma exosome RNA. The expression of these circRNAs also showed significant correlation with both clinical features, serum IgG level and MSG focus scores. Receiver operating characteristic analysis showed that the indices comprised of both the two circRNAs and clinical features were better able to distinguish pSS from non-pSS subjects with high mean areas under the curve of 0.93 in the MSGs and 0.92 in the plasma exosomes. Conclusion This study indicated the potential roles of circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers for the diagnosis of pSS.

Download Full-text

Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat (Avena sativa L.)

10.21203/rs.3.rs-20962/v3 ◽

2020 ◽

Author(s):

Zheng Yang ◽

Kai Wang ◽

Usman Aziz ◽

Cuizhu Zhao ◽

Meng Zhang

Keyword(s):

Real Time ◽

Avena Sativa ◽

Real Time Pcr ◽

Reference Genes ◽

Expression Patterns ◽

Single Copy ◽

Initiation Factor ◽

Pcr Analysis ◽

Systematic Research ◽

Quantitative Real Time Pcr

Abstract Background: Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, no suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids.Results: In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for “all samples”, “shoots and roots of seedlings”, “developing seeds” and “developing endosperms” were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively. Among these RGs, UBC21 was a four-copy duplicated gene. The reliability was validated by the expression patterns of four various genes normalized to the most and the least stable RGs in different sample sets.Conclusions: Results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat.

Download Full-text

Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum based on transcriptome data

BMC Plant Biology ◽

10.1186/s12870-019-2108-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Xiaowei Wang ◽

Zhijun Wu ◽

Wenqi Bao ◽

Hongyan Hu ◽

Mo Chen ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Stress Responses ◽

Real Time Pcr ◽

Abiotic Stresses ◽

Reference Genes ◽

Expression Patterns ◽

Polygonum Cuspidatum ◽

Quantitative Real Time Pcr ◽

Different Tissues

Abstract Background Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data. Results Twelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes. Conclusions We propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.

Download Full-text

Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat (Avena sativa L.)

Plant Methods ◽

10.1186/s13007-020-00679-1 ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Zheng Yang ◽

Kai Wang ◽

Usman Aziz ◽

Cuizhu Zhao ◽

Meng Zhang

Keyword(s):

Real Time ◽

Avena Sativa ◽

Real Time Pcr ◽

Reference Genes ◽

Expression Patterns ◽

Single Copy ◽

Initiation Factor ◽

Pcr Analysis ◽

Systematic Research ◽

Quantitative Real Time Pcr

Abstract Background Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, no suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids. Results In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for “all samples”, “shoots and roots of seedlings”, “developing seeds” and “developing endosperms” were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively. Among these RGs, UBC21 was a four-copy duplicated gene. The reliability was validated by the expression patterns of four various genes normalized to the most and the least stable RGs in different sample sets. Conclusions Results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat.

Download Full-text

Identification of tRNA-derived fragments and their potential roles in diabetic cataract rats

Epigenomics ◽

10.2217/epi-2020-0193 ◽

2020 ◽

Vol 12 (16) ◽

pp. 1405-1418

Author(s):

Xiaoyan Han ◽

Lei Cai ◽

Yi Lu ◽

Dan Li ◽

Jin Yang

Keyword(s):

Real Time ◽

Rna Sequencing ◽

Small Rna ◽

Real Time Pcr ◽

Bioinformatics Analysis ◽

Bioinformatic Analysis ◽

Transfer Rna ◽

Small Rna Sequencing ◽

Quantitative Real Time Pcr ◽

Diabetic Cataract

Aim: To illustrate the expression profile of transfer RNA-derived fragments and reveal their putative role in the pathogenesis of diabetic cataract (DC) rats. Materials & methods: Small RNA sequencing was conducted in the lens epithelium of rats lens. The data were validated by quantitative real-time PCR, and bioinformatic analysis was performed to explore the roles of the fragments in DC pathogenesis. Results: A total of 213 differentially expressed tRNA-related fragments were identified, in which 111 were upregulated and 102 were downregulated in DC rats. Bioinformatics analysis revealed that several associated pathways might participate in the development of DC rats. Conclusion: tRNA-derived fragments may be involved in the pathogenesis of DC rats.

Download Full-text

Expression patterns of HMGA2 in the placenta during pregnancy

10.1101/2020.12.07.20245092 ◽

2020 ◽

Author(s):

Lars Burchardt ◽

Andrea Gottlieb ◽

Burkhard M. Helmke ◽

Werner Wosniok ◽

Wolfgang Kuepker ◽

...

Keyword(s):

Real Time ◽

Gestational Age ◽

Real Time Pcr ◽

Expression Patterns ◽

Critical Role ◽

First Trimester ◽

Postnatal Life ◽

Invasive Growth ◽

Quantitative Real Time Pcr ◽

Mesenchymal Transition

AbstractBackgroundHigh-mobility group AT-hook 2 (HMGA2) expression can be detected in many embryonic and fetal tissues but becomes down-regulated during postnatal life except for many benign and malignant tumors. In the latter case, its expression has been correlated with epithelial-mesenchymal transition and invasive growth. The placenta contributes essentially to proper development of the embryo and the fetus. In a tumor-like manner it shows rapid invasive growth during the first weeks of gestation. To address the possible role of HMGA2 during placental development, we have measured its expression throughout the prenatal period and in term placentae by mRNA quantification as well as by immunohistochemistry.MethodsExpression of HMGA2 and HPRT was measured on 89 fetal placentas, encompassing calendar gestational age of five to 41 weeks, using quantitative real time-PCR. In eleven cases in addition immunohistochemistry was used to determine the localization of HMGA2 and to compare with data obtained by quantitative real time-PCR.ResultsThe expression of HMGA2 was found to be inversely correlated with gestational age (p < 0.001). For the first part of the first trimester the level of HMGA2 is high. After that the expression shows a decline down to a baseline level where it remains until birth. HMGA2 protein was mainly detected in the nuclei of the stromal cells in the placental villi.ConclusionsDuring pregnancy, the expression of HMGA2 follows a non-linear pattern of decrease. In the first trimester, from two to three weeks after the implantation of the conceptus until the blood supply is established (hypoxic phase), the expression is high, indicating a critical role during early development and in the control of its invasive behavior, respectively.

Download Full-text