scholarly journals Selection of appropriate reference genes for quantitative real-time PCR in Clerodendrum trichotomum

2019 ◽  
Author(s):  
Yajie Hua ◽  
Yuanzheng Yue ◽  
Gongwei Chen ◽  
Taotao Yan ◽  
Wenjie Ding ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Quantitative Real Time Pcr ◽  
Mechanism Research ◽  
The Stability ◽  
Suitable Reference ◽  
Important Medicinal Plant ◽  
Selection Of

AbstrsctClerodendrum trichotomum, an important medicinal plant, has excellent salt tolerance and beautiful ornamental character. However, reliable reference genes for quantitative real-time PCR data (qRT-PCR) in C. trichotomum have not been investigated. Using our previous transcriptome data, 17 reference genes were selected in different tissues (leaves, flowers, fruits, stems, and roots) and under various abiotic stresses (salt, drought, flood, and heat) for C. trichotomum, using four different reference gene analysis software types: GeNorm, NormFinder, BestKeeper and ReFinder. The results identified RPL, ACT and HSP70 as the three most suitable reference genes for tissues. Genes ACT and AP-2 were most stably expressed under drought stress; MDH and UBCE2 were stable under flooding stress; RPL and UBCE2 were most stable under salt stress; and MDH and EF-1A were most appropriate under heat stress. For abiotic treatments, RPL, MDH and AP-2 were the most stable reference genes; and AP-2, RPL and ACT were stably expressed in all examined samples. The expression profile of the genes for Na+/H+ Exchanger1 (ClNHX1) and laccase (ClLAC) were selected to validate the stability of the determined reference genes. Our study provided reliable normalization for gene expression analysis and ensured more accurate data for further molecular mechanism research in C. trichotomum.

scholarly journals Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

2018 ◽  
Vol 19 (8) ◽  
pp. 2258 ◽  
Author(s):  
Yuning Hu ◽  
Hongtuo Fu ◽  
Hui Qiao ◽  
Shengming Sun ◽  
Wenyi Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Translation Initiation Factor ◽  
Suitable Reference Gene ◽  
Quantitative Real Time Pcr ◽  
Macrobrachium Nipponense ◽  
The Stability ◽  
Suitable Reference

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

scholarly journals Evaluation of Reference Genes and Normalization Strategy for Quantitative Real-Time PCR in Human Pancreatic Carcinoma

2012 ◽  
Vol 32 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Beatrice Mohelnikova-Duchonova ◽  
Martin Oliverius ◽  
Eva Honsova ◽  
Pavel Soucek
Keyword(s):  
Pancreatic Carcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Rna Degradation ◽  
Pancreatic Tumors ◽  
Quantitative Real Time Pcr ◽  
Expression Studies ◽  
The Stability ◽  
Suitable Reference

Histologically verified pairs (n= 10) of pancreatic tumors and non-neoplastic tissues were used for quantitative real-time PCR and the stability of 24 reference genes was analyzed with geNorm and NormFinder software. Raw Cqvalues correlated with the degree of RNA degradation. This correlation was abolished by normalization to Cqof 18S endogenous control gene. Both geNorm and NormFinder programs suggested EIF2B1, ELF1, MRPL19, and POP4 as the same most stable genes. We have thus identified suitable reference genes for future expression studies in pancreatic carcinoma. Normalization method reducing the effects of RNA degradation on the quality of results was also developed.

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