scholarly journals Expression Stability of Internal Reference Gene in Response to Trichoderma polysporum Infection in Avena fatua L.

2021 ◽  
Author(s):  
Haixia Zhu ◽  
Yongqiang Ma ◽  
Liang Cheng
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Avena Fatua ◽  
Expression Stability ◽  
Internal Reference ◽  
Qpcr Analysis ◽  
Suitable Combination

Abstract In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .

scholarly journals Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Zhaoping Yan ◽  
Jinhang Gao ◽  
Xiuhe Lv ◽  
Wenjuan Yang ◽  
Shilei Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Pancreatitis ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Expression Stability ◽  
Rt Pcr ◽  
Comprehensive Method ◽  
Suitable Combination ◽  
Suitable Reference

The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α> 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.

scholarly journals Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2428 ◽  
Author(s):  
Guanglin Niu ◽  
Yalan Yang ◽  
YuanYuan Zhang ◽  
Chaoju Hua ◽  
Zishuai Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Muscle Development ◽  
Expression Stability ◽  
Skeletal Muscle Development ◽  
Expression Levels ◽  
Suitable Reference

The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes (ACTB,GAPDH,RNF7,RHOA,RPS18,RPL32,PPIA,H3F3,API5,B2M,AP1S1,DRAP1,TBP,WSB, andVAPB) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated thatGAPDHandACTBhad the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development.RPS18,API5, andVAPBhad stable expression levels in prenatal stages, whereasAPI5,RPS18,RPL32, andH3F3had stable expression levels in postnatal stages.API5andH3F3expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.

scholarly journals Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

2021 ◽  
Vol 12 ◽  
Author(s):  
Gothandapani Sellamuthu ◽  
Shan Amin ◽  
Jan Bílý ◽  
Jirí Synek ◽  
Roman Modlinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Gene Selection ◽  
Experimental Conditions ◽  
Tissue Specific ◽  
Reference Gene Selection ◽  
Ips Sexdentatus

Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.

scholarly journals Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 451 ◽  
Author(s):  
Junchao Zhang ◽  
Wengang Xie ◽  
Xinxuan Yu ◽  
Zongyu Zhang ◽  
Yongqiang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Expression Stability ◽  
Forage Production ◽  
Experimental Conditions ◽  
Elymus Sibiricus ◽  
Suitable Reference ◽  
Selection Of

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

scholarly journals Validation of reference genes for expression analysis in a murine trauma model combining traumatic brain injury and femoral fracture

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ellen Otto ◽  
Paul Köhli ◽  
Jessika Appelt ◽  
Stefanie Menzel ◽  
Melanie Fuchs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Reference Gene ◽  
Expression Analysis ◽  
Bone Fracture ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Tissue Specific ◽  
Qrt Pcr

Abstract Systemic and local posttraumatic responses are often monitored on mRNA expression level using quantitative real-time PCR (qRT-PCR), which requires normalisation to adjust for confounding sources of variability. Normalisation requests reference (housekeeping) genes stable throughout time and divergent experimental conditions in the tissue of interest, which are crucial for a reliable and reproducible gene expression analysis. Although previous animal studies analysed reference genes following isolated trauma, this multiple-trauma gene expression analysis provides a notable study analysing reference genes in primarily affected (i.e. bone/fracture callus and hypothalamus) and secondarily affected organs (i.e. white adipose tissue, liver, muscle and spleen), following experimental long bone fracture and traumatic brain injury. We considered tissue-specific and commonly used top-ranked reference candidates from different functional groups that were evaluated applying the established expression stability analysis tools NormFinder, GeNorm, BestKeeper and RefFinder. In conclusion, reference gene expression in primary organs is highly time point as well as tissue-specific, and therefore requires careful evaluation for qRT-PCR analysis. Furthermore, the general application of Ppia, particularly in combination with a second reference gene, is strongly recommended for the analysis of systemic effects in the case of indirect trauma affecting secondary organs through local and systemic pathophysiological responses.

scholarly journals Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

2021 ◽  
Author(s):  
Zhongyi Yang ◽  
Rui Zhang ◽  
Zhichun Zhou
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Geometric Mean ◽  
Gene Transcript ◽  
Expression Stability ◽  
Schima Superba ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Different Tissues

Abstract Background Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

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