Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: The balance between accuracy and precision

Nordgård, Oddmund; Kvaløy, Jan Terje; Farmen, Ragne Kristin; Heikkilä, Reino

doi:10.1016/j.ab.2006.06.020

Cited by 89 publications

(73 citation statements)

References 28 publications

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“…Transcriptional expressions of the selected target genes (fadD1, fadD2, fadA, fabH1, fabG, rhlA, and rhlB) were evaluated by the quantification of mRNA transcripts as described previously (22). Briefly, 500-l samples were taken from each treatment at 8,20,24,30, and 48 h, mixed with 1 ml of RNAprotect bacterial reagent (Qiagen, Valencia, CA), and stored at Ϫ20°C. RNA was extracted using the RNeasy minikit (Qiagen, Valencia, CA) from extraction volumes that were standardized at a cell number of 1 ϫ 10 9 CFU.…”

Section: Methodsmentioning

confidence: 99%

“…The relative expression ratio (RER) of each target gene (T) was calculated from control (glucose) and treatment (C 18 or C 18 -d 35 ) C T values of the target and two reference (R1 and R2) genes at each growth stage using the Nordgård model (equation 1) (24). An effect on gene expression was considered significant when the corresponding ratios were Ͼ2 or Ͻ0.5.…”

Section: Methodsmentioning

confidence: 99%

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Fatty Acid Cosubstrates Provide β-Oxidation Precursors for Rhamnolipid Biosynthesis in Pseudomonas aeruginosa, as Evidenced by Isotope Tracing and Gene Expression Assays

Zhang¹,

Veres-Schalnat

Somogyi

et al. 2012

Appl Environ Microbiol

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Rhamnolipids have multiple potential applications as "green" surfactants for industry, remediation, and medicine. As a result, they have been intensively investigated to add to our understanding of their biosynthesis and improve yields. Several studies have noted that the addition of a fatty acid cosubstrate increases rhamnolipid yields, but a metabolic explanation has not been offered, partly because biosynthesis studies to date have used sugar or sugar derivatives as the carbon source. The objective of this study was to investigate the role of fatty acid cosubstrates in improving rhamnolipid biosynthesis. A combination of stable isotope tracing and gene expression assays was used to identify lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis when fatty acid cosubstrates are present. To this end, we compared the rhamnolipids produced and their yields using either glucose alone or glucose and octadecanoic acid-d 35 as cosubstrates. Using a combination of sugar and fatty acids, the rhamnolipid yield was significantly higher (i.e., doubled) than when glucose was used alone. Two patterns of deuterium incorporation (either 1 or 15 deuterium atoms) in a single Rha-C 10 lipid chain were observed for octadecanoic acid-d 35 treatment, indicating that in the presence of a fatty acid cosubstrate, both de novo fatty acid synthesis and ␤-oxidation are used to provide lipid precursors for rhamnolipids. Gene expression assays showed a 200-to 600-fold increase in the expression of rhlA and rhlB rhamnolipid biosynthesis genes and a more modest increase of 3-to 4-fold of the fadA ␤-oxidation pathway gene when octadecanoic acid was present. Taken together, these results suggest that the simultaneous use of de novo fatty acid synthesis and ␤-oxidation pathways allows for higher production of lipid precursors, resulting in increased rhamnolipid yields.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Fatty Acid Cosubstrates Provide β-Oxidation Precursors for Rhamnolipid Biosynthesis in Pseudomonas aeruginosa, as Evidenced by Isotope Tracing and Gene Expression Assays

Zhang¹,

Veres-Schalnat

Somogyi

et al. 2012

Appl Environ Microbiol

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“…Amplifi cation curves were analyzed using LinReg software v12.7 (Ramakers et al, 2003) to correct for baseline values that produce much variability in amplifi cation effi ciency (Ruijter et al, 2009). The amplifi cation effi ciency was assumed to be the same for all samples per amplicon and PCR run, as the observed variability has been shown to refl ect random error rather than a true variation (Nordgard et al, 2006). The relative gene expression (RGE) was determined by normalizing the effi ciency-corrected expression levels of the target genes to that of 18S rRNA, a commonly-used reference gene in multiple studies involving cartilage and meniscus (Upton et al, 2003;Hofstaetter et al, 2004;Valiyaveettil et al, 2005;Zielinska et al, 2009).…”

Section: Me Levenston Et Al Discrimination Of Meniscal Cell Phenotypesmentioning

confidence: 99%

Discrimination of meniscal cell phenotypes using gene expression profiles

Son

Levenston²

2012

eCM

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The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fi brocartilage tissue engineering. Although functional assessment of the fi nal resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifi able characteristics of meniscal cells and thereby fi nd phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus). The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifi able metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fi brocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defi ning the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

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“…Primer sequences can be found in the supplement (Supplemental Table 3). Standard deviation for relative fold changes was calculated using error propagation (Nordgård et al 2006).…”

Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments

Zagalak

Menzi

Schmich

et al. 2015

RNA

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Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective downregulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.

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Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: The balance between accuracy and precision

Cited by 89 publications

References 28 publications

Fatty Acid Cosubstrates Provide β-Oxidation Precursors for Rhamnolipid Biosynthesis in Pseudomonas aeruginosa, as Evidenced by Isotope Tracing and Gene Expression Assays

Fatty Acid Cosubstrates Provide β-Oxidation Precursors for Rhamnolipid Biosynthesis in Pseudomonas aeruginosa, as Evidenced by Isotope Tracing and Gene Expression Assays

Discrimination of meniscal cell phenotypes using gene expression profiles

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments

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