Evaluation of appropriate reference gene for normalization of microRNA expression by real-time PCR in Lablab purpureus under abiotic stress conditions
Abstract
MicroRNAs (miRNAs) play key roles in plant responses to biotic and abiotic stresses by modulating their own expression and a wide array of target mRNAs. Reverse transcription quantitative real-time PCR is a sensitive and widely used method to study miRNA expression profile. Accurate analysis and interpretation of results require the selection of an appropriate reference gene. Reference genes selected should have a constant expression level under different stress conditions. Six reference candidates, including two miRNAs (miRNA 156 and miRNA 172), an rRNA (5S rRNA), a snRNA (U6) and two protein coding genes (actin and protein phosphatase 2A), were selected for normalization of miRNA expression in Lablab purpureus. The expression stability of candidate reference genes was investigated in ten samples and analysed using NormFinder, BestKeeper and hkgFinder softwares. The analyses suggested that miRNA 156 is the appropriate reference gene, as it had better expression stability than protein-coding genes, and other non-coding RNAs.
Acknowledgements
Adikeshavan Thilagavathy acknowledges the Department of Science and Technology (DST), New Delhi, India, for INSPIRE Fellowship (Code No: IF120387). The authors acknowledge Department of Biochemistry, Indian Institute of Science, for providing Central instrumentation facility to carry out RT-qPCR using Biorad iQ5 Multicolor Real Time PCR Detection System.
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- Abbreviations
- Cq
quantification cycle
- miRNA
microRNA
- PP2A
protein phosphatase 2A
- RT-qPCR
reverse transcription quantitative PCR
- SD
standard deviation.
©2016 Institute of Molecular Biology, Slovak Academy of Sciences
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- Cellular and Molecular Biology
- Molecular detection of Mycobacterium tuberculosis complex in the 8th century skeletal remains from the territory of Slovakia
- Cellular and Molecular Biology
- First report of microorganisms of Caucasus glaciers (Georgia)
- Cellular and Molecular Biology
- Codon optimization of Aspergillus niger feruloyl esterase and its expression in Pichia pastoris
- Botany
- Response of lichens Cladonia arbuscula subsp. mitis and Cladonia furcata to nitrogen excess
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- No confirmation for previously suggested presence of diploid cytotypes of Sesleria (Poaceae) on the Balkan Peninsula
- Botany
- RCD1 homologues and their constituent WWE domain in plants: analysis of conservation through phylogeny methods
- Botany
- Nucleoli migration coupled with cytomixis
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- Evaluation of appropriate reference gene for normalization of microRNA expression by real-time PCR in Lablab purpureus under abiotic stress conditions
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- The fractal nature of the latitudinal biodiversity gradient
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- A new species of Neoribates (Neoribates) (Acari: Oribatida: Parakalummidae) with key to the Neotropical species of the subgenus
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