Designing PCR Primer for DNA Methylation Mapping

Li, Long-Cheng

doi:10.1007/978-1-59745-528-2_19

Cited by 28 publications

(23 citation statements)

References 16 publications

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“…To determine the methylation status of these CpG sites in human CAMP promoter, bisulfite sequencing was performed. Because a product size larger than 300 bp is very difficult to amplify [19], three short fragments containing CpG sites in human CAMP promoter were designed. The CAMP-F1 fragment (185 bp) has 2 CpG sites, CAMP-F2 fragment (172 bp) and CAMP-F3 fragment (179 bp) contains 5 and 2 CpG sites, respectively.…”

Section: Resultsmentioning

confidence: 99%

DNA methylation directly downregulates human cathelicidin antimicrobial peptide gene (CAMP) promoter activity

Chen

Qin

et al. 2017

Oncotarget

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LL-37, the active product of human cathelicidin antimicrobial peptide (CAMP) has a broad spectrum of antibacterial activity. LL-37 also has important physiological functions in immune regulation, angiogenesis and in modulating apoptosis. The roles of LL-37 in oral squamous cell carcinoma (OSCC) are still not clear. The correlation between DNA methylation and human CAMP expression is also unknown. Here human CAMP/LL-37 expression was assessed by immunohistochemistry in normal and OSCC tissues. The results indicated that low expression of CAMP/LL-37 correlated with histological differentiation and lymph node metastasis and also promoted tumor progression. A cell-specific methylation pattern in the promoter region of human CAMP was detected. Treatment with 5-aza-2′-deoxycytidine, a DNA demethylation reagent can increase human CAMP expression in epithelial cancer cells. The reporter assay showed that unmethylated human CAMP promoter activity was significantly higher than methylated promoter activity. Taken together, these results suggested that human CAMP/LL-37 might act as a tumor-suppressor in OSCC and DNA methylation might play roles during carcinogenesis via directly downregulating human CAMP promoter activity.

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Section: Resultsmentioning

confidence: 99%

DNA methylation directly downregulates human cathelicidin antimicrobial peptide gene (CAMP) promoter activity

Chen

Qin

et al. 2017

Oncotarget

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“…The primers used for the methylation-specific PCR (MSP) amplification were designed from published DNA sequences in the 5 0 promoter region of Hoxa10 [45] using the MethPrimer software [46]: Methylated sequence: forward, 5 0 -GGGCGAAAGGGGGCGGGT-3 0 , and reverse, 5 0 -GCGCCCGCCCGCCGCCT-3 0 ; Unmethylated sequence: forward, 5 0 -GGGTGAAAGGGGGCTGGT-3 0 , and reverse, 5 0 -ACACCCACCCAC-CACCT-3 0 . The 140-bp region amplified with these primers includes eight CpG sites.…”

Section: Sodium Bisulfite Dna Modification and Methylationspecific Pcrmentioning

confidence: 99%

“…The primers used for the bisulfite genomic sequencing PCR amplification were designed from mouse DNA sequences that were conserved in the 5 0 promoter region of human HOXA10 gene [47] using the MethPrimer software [46]: forward, 5 0 -TATTTTGAGGTAGTTTTTATAGTTT-3 0 , and reverse, 5 0 -ATAACCCTTTCTAACTAACATT-3 0 . The 271-bp region amplified with these primers includes 20 CpG sites.…”

Section: Bisulphite Sequencing Of Hoxa10 Genementioning

confidence: 99%

Experimental Murine Endometriosis Induces DNA Methylation and Altered Gene Expression in Eutopic Endometrium1

Lee

Taylor

2009

Biology of Reproduction

172

156

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The eutopic endometrium in women with endometriosis demonstrates diminished endometrial receptivity and altered gene expression. It is unknown if the endometrium being defective gives rise to a predisposition toward endometriosis and infertility or, alternatively, if endometriosis causes the altered endometrial receptivity. Here we created experimental endometriosis in mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. Methylation of Hoxa10 was also evaluated as a potential mechanism responsible for altered gene expression. Expression of each gene was measured using quantitative real-time RT-PCR at 14 wk after induction of endometriosis. Expression of Hoxa10 and Hoxa11, which are necessary for endometrial receptivity, were decreased in the endometriosis group. Insulin-like growth factor binding protein-1 (Igfbp1) mRNA was decreased in the endometriosis group. However, there was no change in Integrin beta3 (Itgb3) mRNA expression. Total progesterone receptor (Pgr-AB) was increased in the endometriosis group and the ratio of Pgr-B to Pgr-AB was increased, indicating a shift from Pgr-A to Pgr-B expression. Basic transcription element-binding protein-1 (Bteb1), official symbol and name Klf9, Kruppel-like factor 9, which functionally interacts with Pgr in endometrium, was also decreased in the endometriosis group. In addition, hypermethylation of Hoxa10 in the endometriosis group was shown by methylation-specific PCR and confirmed by bisulfite sequencing. These findings demonstrate that normal endometrium, when placed in an ectopic location to create experimental endometriosis, led to characteristic changes in gene expression in eutopic endometrium. These data suggest the existence of a signal conduction pathway from endometriosis that alters endometrial gene expression through altered Pgr signaling and epigenetic programming.

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“…Bisulfite modification, MSP and BSP were performed as described before [19,20] . The primers of MSP and BSP are shown in Table 1.…”

Section: Methylation-specific Pcr (Msp) and Bisulfitesequencing Pcr (mentioning

confidence: 99%

Methylation of PTCH1a gene in a subset of gastric cancers

Du¹,

Ye²,

Gao³

et al. 2009

WJG

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RESULTS: Methylation of PTCH1a TRR was observedin AGS cells and a subset of gastric cancer tissues (32%, 55/170), while no methylation amplification products were observed in any normal tissues by MSP. The methylation of PTCH1a TRR was correlated negatively with PTCH1 expression (Spearman's r = -0.380, P = 0.000). However, methylation of PTCH1a TRR was not related to the gastric cancer patients' clinical features, such as sex, age of onset, clinical stage, lymph node metastasis or histological grade. The methylation of PTCH1a TRR in AGS cells was almost converted to non-methylation after 5-Aza-dC treatment, which increased PTCH1 expression (5.3 ± 2.5 times; n = 3) and apoptosis rate (3.0 ± 0.26 times; P < 0.05; n = 3). CONCLUSION:Methylation of PTCH1a TRR is present in a subset of gastric cancers and correlated negatively with PTCH1 expression. This may be an early event in gastric tumorigenesis and a new treatment target.

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Designing PCR Primer for DNA Methylation Mapping

Cited by 28 publications

References 16 publications

DNA methylation directly downregulates human cathelicidin antimicrobial peptide gene (CAMP) promoter activity

DNA methylation directly downregulates human cathelicidin antimicrobial peptide gene (CAMP) promoter activity

Experimental Murine Endometriosis Induces DNA Methylation and Altered Gene Expression in Eutopic Endometrium1

Methylation of PTCH1a gene in a subset of gastric cancers

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