scholarly journals Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3137
Author(s):  
Juan Zhao ◽  
Cheng Wang ◽  
Lin Zhang ◽  
Aiai Lei ◽  
Linjie Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Target Genes ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Stability ◽  
Transport Pathway ◽  
Metabolic Role ◽  
Suitable Reference

As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except GAPT4, indicating that they were stably expressed in the rumen tissues throughout development. RPS4X and RPS6 were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (IGF1 and TOP2A), normalized by the selected most stable RGs (RPS4X and RPS6), were consistent with the results of RNA sequencing, while the use of GAPDH and ACTB as RGs resulted in altered profiles. Overall, RPS4X and RPS6 showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.

scholarly journals Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaoli Tang ◽  
Hongyan Wang ◽  
Chuyang Shao ◽  
Hongbo Shao
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Profiles ◽  
Elongation Factor ◽  
Gene Expression Profiles ◽  
Stable Reference Gene ◽  
Experimental Conditions ◽  
Suitable Reference

Kosteletzkya virginica(L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene inK. virginicawhich showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA),β-actin (ACT),α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and18SrRNAwas assessed to be the most stable reference gene in this study. However,TUAwas identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies inK. virginica.

scholarly journals Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3260 ◽  
Author(s):  
Kai Wang ◽  
Yi Niu ◽  
Qijun Wang ◽  
Haili Liu ◽  
Yi Jin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Dynamic Range ◽  
Expression Profiles ◽  
Mrna Levels ◽  
Expression Stability ◽  
Degenerate Primers ◽  
Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

scholarly journals RNAseq Studies Reveal Distinct Transcriptional Response to Vitamin a (VA) Deficiency in Small Intestine (SI) versus Colon, Discovering Novel VA-regulated Genes (P15-002-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Zhi Chai ◽  
Yafei Lyu ◽  
Qiuyan Chen ◽  
Cheng-Hsin Wei ◽  
Lindsay Snyder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Vitamin A ◽  
Target Genes ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Differentially Expressed Gene ◽  
Gene Expression Profiles ◽  
Illumina Hiseq ◽  
The Impact

Abstract Objectives To characterize and compare the impact of vitamin A (VA) deficiency on gene expression patterns in the small intestine (SI) and the colon, and to discover novel target genes in VA-related biological pathways. Methods vitamin A deficient (VAD) mice were generated by feeding VAD diet to pregnant C57/BL6 dams and their post-weaning offspring. Total mRNA extracted from SI and colon were sequenced using Illumina HiSeq 2500 platform. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression patterns and co-expression patterns. Results The comparison between vitamin A sufficient (VAS) and VAD groups detected 49 and 94 DEGs in SI and colon, respectively. According to GO information, DEGs in the SI demonstrated significant enrichment in categories relevant to retinoid metabolic process, molecule binding, and immune function. Immunity related pathways, such as “humoral immune response” and “complement activation,” were positively associated with VA in SI. On the contrary, in colon, “cell division” was the only enriched category and was negatively associated with VA. WGCNA identified modules significantly correlated with VA status in SI and in colon. One of those modules contained five known retinoic acid targets. Therefore we have prioritized the other module members (e.g., Mbl2, Mmp9, Mmp13, Cxcl14 and Pkd1l2) to be investigated as candidate genes regulated by VA. Comparison of co-expression modules between SI and colon indicated distinct VA effects on these two organs. Conclusions The results show that VA deficiency alters the gene expression profiles in SI and colon quite differently. Some immune-related genes (Mbl2, Mmp9, Mmp13, Cxcl14 and Pkd1l2) may be novel targets under the control of VA in SI. Funding Sources NIH training grant and NIH research grant. Supporting Tables, Images and/or Graphs

scholarly journals PCA-based unsupervised feature extraction for gene expression analysis of COVID-19 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kota Fujisawa ◽  
Mamoru Shimo ◽  
Y.-H. Taguchi ◽  
Shinya Ikematsu ◽  
Ryota Miyata
Keyword(s):  
Gene Expression ◽  
Feature Extraction ◽  
Target Genes ◽  
Gene Selection ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Principal Component ◽  
Data Set ◽  
Immune Related Genes ◽  
Unsupervised Feature Extraction

AbstractCoronavirus disease 2019 (COVID-19) is raging worldwide. This potentially fatal infectious disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the complete mechanism of COVID-19 is not well understood. Therefore, we analyzed gene expression profiles of COVID-19 patients to identify disease-related genes through an innovative machine learning method that enables a data-driven strategy for gene selection from a data set with a small number of samples and many candidates. Principal-component-analysis-based unsupervised feature extraction (PCAUFE) was applied to the RNA expression profiles of 16 COVID-19 patients and 18 healthy control subjects. The results identified 123 genes as critical for COVID-19 progression from 60,683 candidate probes, including immune-related genes. The 123 genes were enriched in binding sites for transcription factors NFKB1 and RELA, which are involved in various biological phenomena such as immune response and cell survival: the primary mediator of canonical nuclear factor-kappa B (NF-κB) activity is the heterodimer RelA-p50. The genes were also enriched in histone modification H3K36me3, and they largely overlapped the target genes of NFKB1 and RELA. We found that the overlapping genes were downregulated in COVID-19 patients. These results suggest that canonical NF-κB activity was suppressed by H3K36me3 in COVID-19 patient blood.

scholarly journals Identification and Validation of Novel Reference Genes in Acute Lymphoblastic Leukemia for Droplet Digital PCR

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 376 ◽  
Author(s):  
Vanessa Villegas-Ruíz ◽  
Karina Olmos-Valdez ◽  
Kattia Alejandra Castro-López ◽  
Victoria Estefanía Saucedo-Tepanecatl ◽  
Josselen Carina Ramírez-Chiquito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Reference Genes ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cellular Processes

Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.

scholarly journals Identification of Potentially Therapeutic Target Genes of Hepatocellular Carcinoma

2020 ◽  
Vol 17 (3) ◽  
pp. 1053
Author(s):  
Chengzhang Li ◽  
Jiucheng Xu
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Expression Profiles ◽  
Differential Expression Analysis ◽  
Gene Expression Profiles ◽  
Functional Enrichment ◽  
Ppi Network ◽  
Hub Genes ◽  
Gene Differential Expression ◽  
Oncomine Database

Background: Hepatocellular carcinoma (HCC) is a major threat to public health. However, few effective therapeutic strategies exist. We aimed to identify potentially therapeutic target genes of HCC by analyzing three gene expression profiles. Methods: The gene expression profiles were analyzed with GEO2R, an interactive web tool for gene differential expression analysis, to identify common differentially expressed genes (DEGs). Functional enrichment analyses were then conducted followed by a protein-protein interaction (PPI) network construction with the common DEGs. The PPI network was employed to identify hub genes, and the expression level of the hub genes was validated via data mining the Oncomine database. Survival analysis was carried out to assess the prognosis of hub genes in HCC patients. Results: A total of 51 common up-regulated DEGs and 201 down-regulated DEGs were obtained after gene differential expression analysis of the profiles. Functional enrichment analyses indicated that these common DEGs are linked to a series of cancer events. We finally identified 10 hub genes, six of which (OIP5, ASPM, NUSAP1, UBE2C, CCNA2, and KIF20A) are reported as novel HCC hub genes. Data mining the Oncomine database validated that the hub genes have a significant high level of expression in HCC samples compared normal samples (t-test, p < 0.05). Survival analysis indicated that overexpression of the hub genes is associated with a significant reduction (p < 0.05) in survival time in HCC patients. Conclusions: We identified six novel HCC hub genes that might be therapeutic targets for the development of drugs for some HCC patients.

scholarly journals Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bo Wang ◽  
Huirong Duan ◽  
Peifang Chong ◽  
Shiping Su ◽  
Lishan Shan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Expression Stability ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Nitraria Tangutorum ◽  
Multiple Species ◽  
Suitable Reference

Abstract Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. In this study we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

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