The DNA methylation status of p21CIP1/WAF1 and KLOTHO promoters was determined by MSP as described elsewhere (Rubinek et al., 2012 (link)). MDA-MB-231 cells were treated with either GTPs or SFN or the combinations of both as described earlier. Genomic DNA was extracted at approximately 70–80% confluence using the Qiagen DNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Approximately 1 μg of genomic DNA was subjected to bisulfite conversion using the EpiTect Bisulfite modification kit (Qiagen) according to the manufacturer’s protocol. For MSP analysis, two sets of specific primers were designed (methylated or unmethylated) for the p21CIP1/WAF1 and KLOTHO promoters. The primer sequences and MSP conditions are given in Supplementary Table S2 . PCR amplification was performed for 2 min at 94 °C followed by 35 cycles (30 s at 94 °C; 30 s at TA °C; 30 s at 72 °C) and extension at 72 °C for 2 min using platinum DNA Taq polymerase (Invitrogen). After amplification, PCR products were resolved in 2% agarose gels and visualized by staining with ethidium bromide using ImageQuant LAS4010 chemidocumentation system.
Epitect bisulfite modification kit
Manufactured by Qiagen
Sourced in United Kingdom
The EpiTect Bisulfite modification kit is a laboratory product designed to perform bisulfite conversion of DNA samples. This process is a crucial step in epigenetic analysis, specifically for the investigation of DNA methylation patterns. The kit provides the necessary reagents and protocols to efficiently convert unmethylated cytosine residues to uracil, while leaving methylated cytosines unchanged. This conversion is a fundamental technique used in various epigenetic studies and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy kit, by Qiagen (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna protein isolation kit, by Qiagen (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Jumpstart taq dna polymerase, by Merck Group (1 mentions)
4 protocols using epitect bisulfite modification kit
1
Methylation Status of p21 and KLOTHO
2
Epigenetic Analysis of Hypertension Genes
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Peripheral blood samples of hypertensive patients and normal controls were transferred to 2 ml vacuum tubes with K2EDTA. Genomic DNA was isolated from whole blood samples by using the AllPrep DNA/RNA/Protein isolation kit (Qiagen in Manchester, United Kingdom). A NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA) was used to analyze the amount and purification of DNA samples. Sodium bisulfite modification of DNA samples was done by using the Epi-Tect Bisulfite Modification Kit (Qiagen, Manchester, UK). According to the EpiTect® MS-HRM PCR Handbook (Qiagen, Manchester, UK), primers were designed. The MS-HRM analysis was performed to analyze the promotor methylation status of KLOTHO and ARNTL genes according to the EpiTect® HRM™ PCR Handbook protocol (Rotor-Gene Q, Qiagen). As methylated and unmethylated controls, universal methylated and unmethylated DNA (EpiTect Control DNA Set, Cat No./ID: 59568) were preferred [24 (link)].
3
CHL1 Promoter Methylation and Expression
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CpG islands were predicted using the MethPrimer software. We extracted Genomic DNA from cell lines with bisulfite treatment method using Epitect Bisulfite Modification Kit (Qiagen, Valencia, CA). Bisulfite genomic sequencing (BGS) was performed with CHL1 primers (Forward primer: 5'-TTTTAAATGAAGGAAAGTAAGAAG-3'; Reverse primer: 5'-TCTACTCCCTTCCTAAATTCTAC-3'). The reagent 5-aza-2′-deoxygcitidine (Sigma-Aldrich, St Louis, MO) was used to test if CHL1 expression could be restored by demethylation treatment, it was also employed for the extraction of total RNA (qPCR method) which we used to detect the expression of CHL1.
4
Promoter Methylation Measurement Protocol
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The procedure for measurement of promoter methylation was described previously [26 (link)]. In short, genomic DNA was prepared from 10–15 mg of proximal colon mucosa using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). The DNA (1 μg) was modified via bisulfite treatment using the EpiTect Bisulfite Modification Kit (Qiagen) followed by PCR amplification using 1 cycle at 94 °C (1 min); 33–35 cycles at 94 °C (30 s), 59 °C (30 s), and 72 °C (1 min); and 1 cycle at 72 °C (5 min). Amplification was performed at a volume of 25 μL comprising bisulfite modified genomic DNA (50 ng), 0.4 μL JumpStart Taq DNA polymerase (Sigma-Aldrich), 2.5 μL 10X PCR buffer, 3.5 μL 25 mM MgCl2 (final concentration 3.5 mM), 0.5 μL 10 mM dNTP mix (final concentration 200 μM), forward and reverse primers (1 μL each), and water to a final volume of 25 μL. The PCR products were analyzed on 2% agarose gels and examined by ethidium bromide staining. The size and authenticity of the PCR products were confirmed by a molecular weight analysis and DNA sequencing. The primers (Sigma-Aldrich) used for DNA methylation studies are shown in Table 2 .
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