Optimization of Nonradioisotopic Single Strand Conformation Polymorphism Analysis with a Conventional Minislab Gel Electrophoresis Apparatus

Oto, Michiei; Miyaké, Satoshi; Yuasa, Yasuhito

doi:10.1006/abio.1993.1379

Cited by 116 publications

(50 citation statements)

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“…The allelic band intensity on the gels was detected by non-radioisotopic means with a commercially available silver staining method (Oto et al, 1993). Primers used for amplification of K-RAS exon 1, containing codons 12 and 13, and for the 17q21 region were shown in Table 1 (Supplementary Materials).…”

Section: Real-time Pcrmentioning

confidence: 99%

SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue

et al. 2009

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SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial-mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT-PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). Expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P ¼ 0.047 and P ¼ 0.014, respectively) and NAT lacking SNAI1 expression (P ¼ 0.054 and P ¼ 0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r ¼ 0.39; P ¼ 0.027) and inversely to CDH1 in NAT (r ¼ À0.46; P ¼ 0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VDR.

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Section: Real-time Pcrmentioning

confidence: 99%

SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue

et al. 2009

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“…This reaction mixture was amplified with Ampli Taq Gold (Perkin Elmer, Roche Molecular Systems Inc., Branchburg, NJ, USA): 94°C for 12 min, 35 cycles of 94°C for 30 s, 61°C for 30 s and 72°C for 30 s, followed by incubation at 72°C for 11 min. PCR products were resolved in 6% acrylamide and stained with a non-isotopic silver nitrate method (Oto et al, 1993). PCR-based methylation analysis using restriction enzymes may be subject to variability if the DNA digestions are not complete.…”

Section: Pcr-based Methylation Assaymentioning

confidence: 99%

Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients

et al. 1999

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Summary Hypermethylation of exon 1 of p16 INK4a was examined in tumour and plasma DNA of a series of breast cancer patients. De novo methylation was observed in the tumours of eight patients (23%), and in plasma DNA in five (14%) of these eight patients. Our data show that de novo methylation of exon 1 of p16 INK4a can be demonstrated in plasma DNA of breast cancer patients, a fact that provides additional evidence of the tumour-related origin of free plasma DNA in cancer patients.

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“…The alleles were separated by mixing 25 µl of the PCR products with a 10-µl volume of loading buffer (total volume 35 µl), 0.02% xylene cyanol and 0.02% bromophenol blue. Electrophoresis was run on non-denaturing 8-12% polyacrylamide gels for 12-15 h at 500 V. After gel electrophoresis, the allelic band intensity was detected by a nonradioisotopic technique using a commercially available silver staining method (Oto et al, 1993).…”

Section: Pcr Conditions Primers and Loh Assaymentioning

confidence: 99%

Detection of loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 and BRCA2 loci in breast cancer: pathological correlations

et al. 1999

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Summary Loss of heterozygosity (LOH) in loci of the 15q15.1, 12p13, 1p32, 17q21 and 13q12-13 regions may collaborate in the inactivatio n of RAD51, RAD52, RAD54, BRCA1, BRCA2 and possibly other genes implicated in the repair of double-stranded DNA and in DNA recombination. We investigate allelic losses in microsatellites of the RAD51, RAD52, RAD54, BRCA1 and BRCA2 regions, and their correlations with nine pathologic parameters in 127 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using 15 markers of the 15q15.1, 12p13.3, 1p32, 17q21 and 13q12-13 regions. LOH was found in the RAD51 region in 32% of tumours, in t he RAD52 region in 16%, in RAD54 in 20% and in the BRCA1 and BRCA2 regions in 49% and 44% respectivel y. Significant correlations between one or more regions with concomitant LOH and pathologic parameters were observed with respect to age (P = 0.008), oestrogen receptor content (P = 0.03), progesterone receptors (P = 0.003), higher grade (P = 0.001), more advanced stage (P = 0.004) and peritumoural vessel involvement (P < 0.0001). The number of cases in which LOH was observed simultaneously in two or more regions was always higher than expected on the basis of their statistical probabilit y, and curiousl y, the three patients with LOH at five regions concomi tantly were under the age of 30 years. These results suggest that LOH at these regions could be related to breast cance r, and probably to a poo r tumour prognosis .

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Optimization of Nonradioisotopic Single Strand Conformation Polymorphism Analysis with a Conventional Minislab Gel Electrophoresis Apparatus

Cited by 116 publications

References 0 publications

SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue

SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue

Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients

Detection of loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 and BRCA2 loci in breast cancer: pathological correlations

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