Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Lee, Changsoo; Kim, Jaai; Shin, Seung Gu; Hwang, Seokhwan

doi:10.1016/j.jbiotec.2005.11.014

Cited by 570 publications

(420 citation statements)

References 23 publications

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“…As a control for the qPCR method, we also measured the copy number of the ColE1 plasmid pBR322 and of the pSC101-derivative pWSK29. The qPCR method yielded a copy number of 16.5 plasmids/chromosome for pBR322, consistent with a range of 15-20 plasmids per chromosome (Lee et al, 2006;LinChao and Bremer, 1986). In addition, pWSK29 was found to have a copy number of 6.7 plasmids/chromosome, closely matching previous measurements for pSC101 of ∼6 plasmids/ chromosome (Cabello et al, 1976;Hasunuma and Sekiguchi, 1977).…”

Section: Plasmid Copy Number Determinationsupporting

confidence: 61%

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New pSC101-derivative cloning vectors with elevated copy numbers

Peterson

Phillips

2008

Plasmid

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Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (∼240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the E. coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.

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Section: Plasmid Copy Number Determinationsupporting

confidence: 61%

“…blaQf : 5′CTACGATACGGGAGGGCTTA-3′; and blaQr : 5′-ATAAATCTGGAGCCGGTGAG-3′ (Lee et al, 2006). The primers used for probing ffh were: ffh-S : 5′-TAAACTCGGTAAGTTCCTGCGCGA-3′; and ffh-AS : 5′-AGCGCCGCGTTAA CAATATCAACC-3′.…”

Section: Copy Number Determinationmentioning

confidence: 99%

New pSC101-derivative cloning vectors with elevated copy numbers

Peterson

Phillips

2008

Plasmid

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“…The quantity and quality of DNA were assessed using a Nanodrop apparatus (ND1000) (Thermo Fisher Scientific Inc., MA, USA). For qPCR, standard curves were prepared with pooled aliquots of digesta and faecal microbial genomic DNA for the absolute quantification of bacteria (Lee et al, 2006). Genus-and species-specific primers (Supplementary Table S2) for Lactobacilli, Enterobacteriaceae, Bifidobacteria, Bacteriodes and Firmicutes were used for the estimation of gene copy number in the caecal and colonic digesta using qPCR on the ABI 7500 Real-Time PCR System (Applied Biosystems Ltd, Carlsbad, CA, USA) using the method of O'Shea et al (2012).…”

Section: Methodsmentioning

confidence: 99%

Pigs that are divergent in feed efficiency, differ in intestinal enzyme and nutrient transporter gene expression, nutrient digestibility and microbial activity

Vigors

Sweeney

O’Shea

et al. 2016

Animal

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Feed efficiency is an important trait in the future sustainability of pig production, however, the mechanisms involved are not fully elucidated. The objective of this study was to examine nutrient digestibility, organ weights, select bacterial populations, volatile fatty acids (VFA's), enzyme and intestinal nutrient transporter gene expression in a pig population divergent in feed efficiency. Male pigs (n = 75; initial BW 22.4 kg SEM 2.03 kg) were fed a standard finishing diet for 43 days before slaughter to evaluate feed intake and growth for the purpose of calculating residual feed intake (RFI). Phenotypic RFI was calculated as the residuals from a regression model regressing average daily feed intake (ADFI) on average daily gain (ADG) and midtest BW 0.60 (MBW). On day 115, 16 pigs (85 kg SEM 2.8 kg), designated as high RFI (HRFI) and low RFI (LRFI) were slaughtered and digesta was collected to calculate the coefficient of apparent ileal digestibility (CAID), total tract nutrient digestibility (CATTD), microbial populations and VFA's. Intestinal tissue was collected to examine intestinal nutrient transporter and enzyme gene expression. The LRFI pigs had lower ADFI ( P < 0.001), improved feed conversion ratio ( P < 0.001) and an improved RFI value relative to HRFI pigs (0.19 v. −0.14 SEM 0.08; P < 0.001). The LRFI pigs had an increased CAID of gross energy (GE), and an improved CATTD of GE, nitrogen and dry matter compared to HRFI pigs ( P < 0.05). The LRFI pigs had higher relative gene expression levels of fatty acid binding transporter 2 ( FABP2) ( P < 0.01), the sodium/glucose co-transporter 1 ( SGLT1) ( P < 0.05), the glucose transporter GLUT2 ( P < 0.10), and the enzyme sucrase-isomaltase (SI) ( P < 0.05) in the jejunum. The LRFI pigs had increased populations of lactobacillus spp. in the caecum compared with HRFI pigs. In colonic digesta HRFI pigs had increased acetic acid concentrations ( P < 0.05). Differences in nutrient digestibility, intestinal microbial populations and gene expression levels of intestinal nutrient transporters could contribute to the biological processes responsible for feed efficiency in pigs.

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“…Thus, serial 10-fold dilutions from 10 10 to 10 0 copy/µl of the plasmids were used as standards. Plots of the logarithm of the template concentration versus the Ct were graphed, and the PCR efficiency was calculated using the equation: E = 10(-1/slope) (22).…”

Section: Methodsmentioning

confidence: 99%

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

et al. 2012

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Abstract. The aim of this study was to determine the expression of miR-21, miR-31, miR-96 and miR-135b in 52 paired colorectal cancer (CRC) tissues and to analyze the correlation between microRNAs (miRNAs) and clinicopathological features. We developed a quantification method that relies on a standard plot, constructed from known concentrations of standards, in order to measure the number of miRNAs. In addition to this, we analyzed the expression levels of miR-21, miR-31, miR-96 and miR-135b in 52 cases of primary CRC and corresponding normal mucosal tissue using real-time PCR with SYBR-Green I. An independent sample t-test was used to compare the differential expression between tumor tissues and normal mucosal tissues. The Mann-Whitney U and Kruskall-Wallis tests were used to compare the correlation between miRNA expression levels and clinicopathological features. The expression of miR-21, miR-31, miR-96 and miR-135b was upregulated in the CRC tissues compared to normal mucosal tissues (P<0.05). Furthermore, miR-21 and miR-135b were positively correlated with the clinical stage (P=0.048 and P=0.029, respectively), while miR-96 and miR-135b were correlated with liver metastasis (P=0.006 and P=0.013, respectively). Our results suggest that miR-21, miR-31, miR-96 and miR-135b may function in the process of CRC development and progression. miR-135b levels in particular may correlate with the degree of malignancy. IntroductionColorectal cancer (CRC) is the third most common type of malignant neoplasm and the third leading cause of cancer-related mortalities worldwide (1). Conventional pathological diagnosis is traumatic, with the majority of cases being detected in the later stage. Early detection of CRC has puzzled clinicians and scientists for years. Even with annual fecal occult-blood testing, which has decreased the 13-year cumulative mortality rate from CRC by 33% (2), the outcome remains poor in patients with advanced disease. Only CRC diagnosed at an early stage is likely to be cured by surgical resection. Genetic alterations present in CRC, including those in APC, K-ras or p53, do not demonstrate a confirmed correlation between their mutation rate and clinical stage (3). Therefore, a reliable, sensitive and specific molecular diagnostic test for CRC is highly desirable from a clinical perspective.Accumulating evidence suggests that microRNAs (miRNAs) play a crucial role in the tumorigenesis and prognosis of cancer (4-7). miRNAs are non-coding, single-stranded RNAs of 18-25 nucleotides in length, which are able to regulate gene expression by inhibiting translation or decreasing stability of target mRNAs. Over the past few years, interest in the identification, detection and utilization of miRNA molecules has expanded rapidly. Bioinformatic analysis suggests that up to 30% of human genes may be regulated by miRNAs, despite the fact that they only constitute approximately 1% of the human genome (8,9).Changes in miRNA expression have been observed in a variety of human tumors, including breast (5), prostate (6),...

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Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Cited by 570 publications

References 23 publications

New pSC101-derivative cloning vectors with elevated copy numbers

New pSC101-derivative cloning vectors with elevated copy numbers

Pigs that are divergent in feed efficiency, differ in intestinal enzyme and nutrient transporter gene expression, nutrient digestibility and microbial activity

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

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