Real‐Time PCR

Fraga, Dean; Meulia, Tea; Fenster, Steven D.

doi:10.1002/9780470089941.et1003s08

Cited by 37 publications

(62 citation statements)

References 36 publications

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“…Although arbitrary end-point RT-PCR can detect obvious changes in gene expression, it suffers from low precision, and often involves visualization of PCR products at the plateau phase of the reaction, when the data do not accurately reflect the quantity of initial starting mRNA material (407). , generate large datasets that demand major computational investigation to extract meaningful data for relevant genes.…”

Section: Bianco Et Almentioning

confidence: 99%

See 1 more Smart Citation

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

Bianco

Anderson

Forrest

et al. 2014

Thyroid

174

106

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Section: Bianco Et Almentioning

confidence: 99%

“…In general, standard primer design rules apply to RT-qPCR except that amplicon size should be restricted to 50-200 bp with a preferred amplicon size around 100 bp to maximize efficiency (407). When possible, primer pairs should span exonexon junctions in cDNA to avoid amplification of potentially contaminating genomic DNA.…”

Section: Bianco Et Almentioning

confidence: 99%

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

Bianco

Anderson

Forrest

et al. 2014

Thyroid

174

106

View full text Add to dashboard Cite

“…UBIQUITIN5 served as a reference gene for qRT-PCR (Gutierrez et al, 2008). Amplification efficiencies were determined using a serial dilution of DNA, and the Pfaffl method was used to calculate fold changes in gene expression relative to the wild-type heart stage (Pfaffl, 2001;Fraga et al, 2008).…”

Section: Genotyping Rna Isolation and Rt-pcr Analysismentioning

confidence: 99%

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage

et al. 2015

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Plants invest a lot of their resources into the production of an extracellular matrix built of polysaccharides. While the composition of the cell wall is relatively well characterized, the functions of the individual polymers and the enzymes that catalyze their biosynthesis remain poorly understood. We exploited the Arabidopsis (Arabidopsis thaliana) seed coat epidermis (SCE) to study cell wall synthesis. SCE cells produce mucilage, a specialized secondary wall that is rich in pectin, at a precise stage of development. A coexpression search for MUCILAGE-RELATED (MUCI) genes identified MUCI10 as a key determinant of mucilage properties. MUCI10 is closely related to a fenugreek (Trigonella foenumgraecum) enzyme that has in vitro galactomannan a-1,6-galactosyltransferase activity. Our detailed analysis of the muci10 mutants demonstrates that mucilage contains highly branched galactoglucomannan (GGM) rather than unbranched glucomannan. MUCI10 likely decorates glucomannan, synthesized by CELLULOSE SYNTHASE-LIKE A2, with galactose residues in vivo. The degree of galactosylation is essential for the synthesis of the GGM backbone, the structure of cellulose, mucilage density, as well as the adherence of pectin. We propose that GGM scaffolds control mucilage architecture along with cellulosic rays and show that Arabidopsis SCE cells represent an excellent model in which to study the synthesis and function of GGM. Arabidopsis natural varieties with defects similar to muci10 mutants may reveal additional genes involved in GGM synthesis. Since GGM is the most abundant hemicellulose in the secondary walls of gymnosperms, understanding its biosynthesis may facilitate improvements in the production of valuable commodities from softwoods.

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“…PCR utilizes a pair of primers, each hybridizing to one strand of a double-stranded DNA target, with the pair spanning a region that is exponentially reproduced. The hybridized primer creates a complimentary strand through a sequential addition of deoxynucleotides [2,3]. This process is summarized in the following steps (i) at > 90°C, the double-stranded DNA is separated (ii) at 50-75°C primer annealing occurs, and (iii) at 72-78°C optimal extension is achieved as depicted in Fig.…”

Section: Introductionmentioning

confidence: 99%

“…This process is summarized in the following steps (i) at > 90°C, the double-stranded DNA is separated (ii) at 50-75°C primer annealing occurs, and (iii) at 72-78°C optimal extension is achieved as depicted in Fig. 1 [2][3][4]. Some of the gold standards such as cell culture and serological assays have been displaced by the introduction of conventional PCR which is used to obtain quantitative data with promising results [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

Real-Time PCR and Real-Time RT-PCR Applications in Food Labelling and Gene Expression Studies

Kashani¹,

Malau‐Aduli²

2014

IJGG

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Abstract:Polymerase chain reaction (PCR) as a scientific invention, has revolutionized molecular biology and led to real-time PCR and later, real-time reverse transcription PCR (Real-Time RT-PCR). These two techniques enable scientists to conduct PCR detection of amplified gene products and expression analysis of targeted genes. Quantitative polymerase chain reaction (qPCR), also called real-time polymerase chain reaction, is a recent modification to PCR that utilizes fluorescent reporter molecular techniques to monitor the production of amplified products during each cycle of the PCR reaction, and enables both detection and quantification of specific sequences in complex mixtures. Over the past decade, real-time PCR applications have rapidly changed the nature of molecular science and become widely used tools in molecular genetics research. Real-time PCR permits specific, sensitive and reproducible manipulation of nucleic acids by combining the nucleic acid amplification and detection steps using gel electrophoresis. Hence, it almost eliminates the need for DNA sequencing or Southern blotting for amplicon identification. One of the many versions of PCR is real-time RT-PCR which has become one of the most broadly used gene amplification and expression methods in molecular biology research. Real-time RT-PCR is commonly employed to discover RNA expression levels through the creation of complimentary DNA (cDNA) transcripts from RNA, and it is frequently confused with real-time PCR. Food labelling provides very important information to help both producers and consumers to make informed choices about healthier and safer food. The process that information from a gene is used in the synthesis of a functional gene product is called gene expression. It enables scientists decipher the functions of genes. Food labelling and gene expression are fundamental to studying the relationships between the human genome, nutrition and health in a relatively new specialist field called nutritional genomics. Nutritional genomics is expected to revolutionize the way health professionals and dieticians treat people in the future. Thus, it is anticipated that the focus of nutritional genomics research will in the future, shift to determining the right type of food for an individual based on his or her genomic compatibility and therefore aid in avoiding foods that are an inappropriate match and could potentially impact negatively on the individual's health. This paper reviews the importance and power of real-time PCR application in food labelling and nutritional genomics, types of fluorescentbased chemistry procedures developed for real-time PCR detection, real-time RT-PCR application in gene expression studies and the great potential of combining these technologies for animal molecular genetics research in sheep and fish.

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Real‐Time PCR

Cited by 37 publications

References 36 publications

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage

Real-Time PCR and Real-Time RT-PCR Applications in Food Labelling and Gene Expression Studies

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