Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane

Quantitative real-time polymerase chain reaction (qPCR) is an essential tool for understanding animal cell’s response to developmental progression or to different experimental conditions at gene expression level. However the reliability of this method heavily lies on proper normalization (measuring a target and a reference gene’s expression from the same sample to correct for technical related variations).Our literature review aimed to summarize the articles addressing the most important livestock species in regards of reference gene stability used as normalizers for quantitative real-time polymerase chain reaction experiments. Stably expressing reference genes were categorized into 14 distinct groups according to gene function. The number of reference genes tested and the publication numbers according to years and the ranking algorithms were also noted.Counting showed that genes encoding ribosomal protein components are ranked as most stable in majority of cases and therefore should be taking into account for qPCR stable normalizer gene finding experiments.

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Optimized QRT-PCR Approach for the Measurable Impact of Adjuvant Cholecalciferol Therapy in Ameliorating Cytokine Gene Expression

European Journal of Medical and Health Sciences ◽

10.24018/ejmed.2021.3.6.1117 ◽

2021 ◽

Vol 3 (6) ◽

pp. 44-50

Author(s):

Javed Akram ◽

Akram Tariq ◽

Gibran Ali ◽

Fridoon Jawad Ahmed ◽

Syeda Saba Aslam

Keyword(s):

Gene Expression ◽

Vitamin D ◽

Polymerase Chain Reaction ◽

Real Time ◽

Polymerase Chain Reaction Analysis ◽

Cytokine Gene Expression ◽

Cytokine Gene ◽

Chain Reaction ◽

Open Label ◽

Polymerase Chain

The endemic Vitamin D deficiency in Pakistan and the current COVID-19 epidemic have converged into a double whammy scenario in Pakistan [1]. Nutritional epigenomic studies have highlighted Vitamin D as a master Vitamin influencing various genomic expressions through its active metabolite 1α,25-dihydroxyvitamin D3 [2]. The objective of this study was to evaluate the measurable impact of adjuvant Cholecalciferol therapy in the Cytokine gene expression of COVID-19 patients by quantitative Real-Time Polymerase Chain Reaction analysis. The trial was a randomized control prospective open label interventional trial done on moderate to severe COVID-19 patients with deranged inflammatory and coagulation biomarkers. SunnyD STAT (Vitamin D3 200000 IU) softgels were given at Day 1, Day 3 and Day 5 of the treatment. Optimized quantitative Real-Time Polymerase Chain Reaction analysis showed decreased genetic expressions of Interleukin 6 (IL-6), Interleukin 2RA (IL-2RA) and Tumor Necrosis Factor (TNF-a) in the interventional group against the age and co-morbidities matched controls, providing molecular and genetic level evidence for the purported mechanism of amelioration of Cytokines induced pathogenic inflammation. However, inherent limitations of the design restrict the generalizability of the results and warrants caution for extrapolation. We recommend randomized placebo-controlled trials with larger sampling and genome wide profiling to infer more definite interpretations.

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Metabolic Adaptations in Skeletal Muscle during Lactation: Complementary Deoxyribonucleic Acid Microarray and Real-Time Polymerase Chain Reaction Analysis of Gene Expression

Endocrinology ◽

10.1210/en.2004-0721 ◽

2004 ◽

Vol 145 (11) ◽

pp. 5344-5354 ◽

Cited By ~ 23

Author(s):

Xiao Qiu Xiao ◽

Kevin L. Grove ◽

M. Susan Smith

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Polymerase Chain Reaction ◽

Real Time ◽

Deoxyribonucleic Acid ◽

Polymerase Chain Reaction Analysis ◽

Chain Reaction ◽

Metabolic Adaptations ◽

Complementary Deoxyribonucleic Acid ◽

Polymerase Chain

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Selection of Reference Genes for Real-time Quantitative Polymerase Chain Reaction Analysis of Light-dependent Anthocyanin Biosynthesis in Chrysanthemum

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.140.1.68 ◽

2015 ◽

Vol 140 (1) ◽

pp. 68-77 ◽

Cited By ~ 7

Author(s):

Yan Hong ◽

SiLan Dai

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Real Time ◽

Reference Genes ◽

Developmental Stages ◽

Anthocyanin Biosynthesis ◽

Light Conditions ◽

Chain Reaction ◽

Horticultural Crops ◽

Polymerase Chain

The light-dependent coloration of the vital organs of horticultural crops affects multiple parts of production and sales. The simplicity of the metabolic pathways of anthocyanins and the characteristics of light-dependent coloration make chrysanthemum (Chrysanthemum ×morifolium) an ideal subject for studying the mechanism of light-regulated anthocyanin biosynthesis. In this study, real-time quantitative reverse transcription–polymerase chain reaction (PCR) was used in the analysis of the expression levels of anthocyanin biosynthesis genes in C. ×morifolium ‘Reagan’. The reference genes selected were those assumed to remain at constant levels in three flower color lines at five floral developmental stages and at two light conditions. Using digital gene expression technology, we selected nine reference genes with moderate expression in the chrysanthemum ray florets at various floral developmental stages under illuminated and dark conditions as the candidate reference genes for further study. After comprehensively analyzing the stability of gene expression with three distinct statistical algorithms, geNorm, NormFinder, and qBase plus, we found that F-box and PP2A were the most stable genes in all of the samples. In addition, we analyzed the relative expression level of the CmF3H gene in different samples to verify the reference genes that we selected. This study provides a consensus list of validated reference genes that will benefit future studies of the expression of chrysanthemum genes involved in anthocyanin biosynthesis and floral development under various light conditions. Moreover, this information will also promote the molecular breeding of horticultural crops for their color modification.

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