DNA extraction from fresh tissues was performed using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Sodium bisulfite conversion was performed using the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (TaKaRa) as per the manufacturer’s instructions. Aberrant DNA-methylation, often reported around the transcription start site (TSS) within a CpG island, was evaluated by Q-MSP. The primer sequences specific for G protein-coupled receptor (GPCR) genes have been shown in Supplementary Table S3 . A standard curve for Q-MSP was constructed by plotting five serially diluted standard solutions of EpiScope Methylated HeLa gDNA (TaKaRa). Normalized methylation value (NMV) for PCR conditions was analyzed as previously described [10 (link),14 (link)]. A standard curve was generated from serial dilutions of EpiScope Methylated HeLa gDNA (TaKaRa). NMV was defined as follows: NMV = (GPCR-S/GPCR-FM)/(ACTB-S/ACTB-FM), where GPCR-S and GPCR-FM represent GPCR methylation level in sample and universally methylated DNA respectively, and ACTB-S and ACTB-FM correspond to β-actin level in sample and universally methylated DNA, respectively.
Episcope methylated hela gdna
Manufactured by Takara Bio
The EpiScope Methylated HeLa gDNA is a genomic DNA product derived from HeLa cells. It is suitable for use as a reference material in epigenetic research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Thermal cycler dice real time system tp800, by Takara Bio (2 mentions)
Methyleasy xceed rapid dna bisulfite modification kit, by Takara Bio (2 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Episcope msp kit, by Takara Bio (1 mentions)
Methyleasy xceed rapid dna bisulphite modification kit, by Takara Bio (1 mentions)
6 protocols using episcope methylated hela gdna
1
Quantitative Methylation Analysis of GPCR Genes
2
Quantitative Methylation Analysis of CpG Sites
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DNA methylation at CpG sites near promoter regions of the target genes was defined via quantitative methylation-specific PCR analysis ( Q-MSP) using the Thermal Cycler Dice Real Time System TP800 (TaKaRa). The sequences of the primers used in this study are presented in Additional File 1: Table S1 . Exon one and CpG sites within views of the promoter region relative to the transcription start site are presented in Additional File 2: Figure S1 18 (link). A standard curve for Q-MSP was constructed by plotting five serially diluted standard solutions of EpiScope Methylated HeLa gDNA (TaKaRa). The normalized methylation value (NMV) was defined as follows: NMV = (GPCRs gene-S/ GPCRs gene-FM)/(ACTB-S/ACTB-FM), where GPCRs gene-S and GPCRs gene-FM represent target gene methylation levels in the tumor sample and universal methylated DNA control, respectively. ACTB-S and ACTB-FM correspond to β-actin (ACTB) in the sample and the universally methylated DNA, respectively.
3
Quantitative Methylation-Specific PCR Analysis
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Aberrant DNA methylation, which often occurs around the transcriptional start site (TSS) within a CpG island, was evaluated using Q-MSP. The sequences of primers used in this study are shown in Supplementary Table S3 . A standard curve for Q-MSP was constructed by plotting five serially diluted standard solutions of EpiScope methylated HeLa gDNA (TaKaRa). To analyse the normalised methylation value (NMV) of the PCR conditions, an analytical method was used as previously described [44 (link)].
4
Quantitative Methylation-Specific PCR for DNA Methylation
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Aberrant DNA methylation, which often occurs around the transcription start site (TSS) within a CpG island, was evaluated by Q-MSP. The sequences of primers used in this study are shown in Additional file 2 : Table S2. Exon one or two and CpG sites within view of the promoter region relative to the TSS are presented in Additional file 3 : Figure S1. A standard curve for Q-MSP was constructed by plotting five serially diluted standard solutions of EpiScope Methylated HeLa gDNA (TaKaRa). The normalized methylation value (NMV) was defined as follows: NMV = (prostanoid receptor gene-S/prostanoid receptor gene-FM)/(ACTB-S/ACTB-FM), where prostanoid receptor gene-S and prostanoid receptor gene-FM represent target gene methylation levels in the tumour sample and in the universal methylated DNA control, respectively. ACTB-S and ACTB-FM correspond to β-actin (ACTB) in the sample and the universally methylated DNA, respectively [15 (link)].
5
Methylation Analysis of HNSCC Samples
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Extraction and bisulfite conversion of genomic DNA from 230 primary HNSCC and 36 noncancerous mucosal samples were performed using the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (TaKaRa, Tokyo, Japan) per the manufacturer’s instructions [12 (link)]. The methylation levels of the CpG islands in the promoters of the SST, TAC1, HCRT, NPY, and GAL genes were determined via Q-MSP with the TaKaRa Thermal Cycler Dice Real Time System TP800 (TaKaRa); the primer sets are listed in Additional file 1 : Table S1. Exon structure and CpG sites within expanded views of the promoter region relative to the transcription start site (TSS) are presented in Additional file 2 : Figure S1. A standard curve was constructed by plotting known concentrations of serially diluted EpiScope Methylated HeLa gDNA (TaKaRa). The normalized methylation value (NMV) was determined as follows: NMV = (Target gene-S/Target gene-FM)/(ACTB-S/ACTB-FM), where Target gene-S and Target gene-FM represent the target gene methylation levels in the tumor sample and universal methylated DNA control, respectively, and ACTB-S and ACTB-FM represent the ACTB (which encodes β-actin) methylation levels in the sample and control, respectively. Analysis was performed using the software (version 1.03A) for the Thermal Cycler Dice Real Time System TP800 (TaKaRa), according to the manufacturer’s directions [13 (link)].
6
Determining CRBN Promoter Methylation in BM-Derived Samples
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Methylation in the CRBN promoter was determined in the BM‐derived genomic DNAs from the 22 post‐Ld samples using methylation‐specific PCR (MSP). DNA modifications were analyzed by bisulphite conversion using the MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (TaKaRa). The MSP analysis was undertaken using the EpiScope MSP kit (TaKaRa). EpiScope Methylated HeLa gDNA (TaKaRa) was used as a positive control. The methylated (CRBN‐M) and unmethylated (CRBN‐UM) primers were designed to detect the CpG island in the CRBN promoter (from transcription starting site +62 to +227). The sequences of the CRBN‐M and CRBN‐UM primers were: 5′‐GCG TAT AAT ATG GGT AAT TAT TTG TC ‐3′ and 5′‐GAT ACG ACC CTA CTA AAC TAA CTC G ‐3′ for CRBN‐M, and 5′‐ TGT ATA ATA TGG GTA ATT ATT TGT TGT ‐3′ and 5′‐ AAT ACA ACC CTA CTA AAC TAA CTC A ‐3′ for CRBN‐UM. We used 6 ng bisulfite‐converted DNA as a template. Polymerase chain reaction was carried out according to the kit manual. Subsequently, agarose gel electrophoresis and ethidium bromide staining were undertaken.
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