L1 promoter methylation analysis was performed on isolated gastrocnemius DNA (described above) using a commercially available methylated DNA immunoprecipitation (MeDIP) kit (Abcam, Cambridge, MA). Prior to performing the MeDIP assay, 1.5 μg of gastrocnemius DNA was digested using MseI due to the fact that this enzyme did not digest DNA within our qPCR primer sequences (New England BioLabs, Ipswich, MA). Following digestion reactions, 1 μg of DNA was used for immunoprecipitation with an anti-5-methylcytosine antibody provided within the kit. qPCR was then performed on the methylated DNA-enriched sample using the L1–3 and L1-Tot primers listed above, given that both primer pairs span CpG-rich areas in the 5′-UTR. Additionally, 0.1 μg of residual input DNA from each sample was used as a control in a parallel reaction to normalize qPCR results. Both the experimental and control wells contained 25 ng of DNA for the reactions and were carried out using the same primer- and SYBR green-based methods as described above for qPCR. Fold change scores in L1 DNA methylation were calculated as follows: 1) 2ΔCq values were calculated whereby ΔCq = input DNA Cq – methylated DNA Cq, and 2) fold change values were then obtained by dividing each individual 2ΔCq value by the SED 2ΔCq group mean.
Medip kit
Manufactured by Abcam
The MeDIP (Methylated DNA Immunoprecipitation) kit is a tool for enriching and detecting methylated DNA fragments from genomic DNA samples. The kit utilizes an antibody specific for 5-methylcytosine to selectively isolate methylated DNA regions, which can then be analyzed using various downstream techniques, such as sequencing or qPCR.
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6 protocols using medip kit
1
L1 Promoter Methylation Analysis in Skeletal Muscle
2
Genome-wide DNA Methylation Analysis
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Genomic DNA was isolated by phenol–chloroform extraction as previously described. The MeDIP assay was carried out as recommended by the methylated DNA immunoprecipitation (MeDIP) kit (Abcam #ab117133). In brief, genomic DNA was sonicated to 200- to 600-bp. A total of 1 μg of the purified DNA fragments was used for the MeDIP reaction. MeDIP were performed with 1 µL of non-immune IgG or 1 µL of 5mC antibody (Abcam). The DNA was released by treatment with proteinase K and further purified for the library DNA preparation.
3
Validating Differential DNA Methylation
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We used a methyl-DNA immunoprecipitation (MeDIP) kit (Abcam) to validate differentially methylated regions (DMRs). After integration of DEGs and DMRs, 2 genes, metallothionein 1 (Mt1) and an organic cation transporter Slc22a13 were selected to study the DMRs via MeDIP. SYBR-green primers were designed for each DMR to have an efficiency near 100% (S1 Table ). MeDIP was performed according to the manufacturer’s protocol. Briefly, genomic DNA was isolated from each rat kidney (n = 5 per group) and sheared for 20 seconds 3x at 25% amplitude to generate DNA fragments of 200–600 bp. Then, 1 ug sheared gDNA was immunoprecipitated with a methylcytosine specific antibody overnight, and the immunoprecipitated was captured by beads, washed, and released from beads by proteinase K digestion. Both MeDIP and an aliquot of sheared gDNA (input DNA) were purified and used for real time PCR. Methylation was expressed as % methylated/input DNA difference and compared to non-pregnant Sprague-Dawley used as the control.
4
MeDIP Isolation of Methylated DNA
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Methylated DNA Immunoprecipitation (MeDIP) Kit (Abcam-117133) was used according to manufacturer’s instructions to isolate methylated DNA from the embryo samples. Special low DNA LoBind tubes (Ep- pendorf #022431021) were used during the MeDIP procedure to prevent loss of DNA. DNA samples were sonicated with 3 pulses of 10–12 s each at level 2 using a Branson Microtip probe, followed by 30–40 s rest on ice between each pulse. Initially, sonicated DNA (25 ng from each pool) was diluted in 450 µL of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and then denatured at 95 °C for 10 min. A portion of the sonicated DNA was reserved as Input (control) and was not subjected to immunoprecipitation. The denatured DNA was placed on ice for 10 min and was then incubated with 5mC monoclonal antibody for 2 h at 4 °C on an orbital shaker (50–100 rpm). After immunoprecipitation, methylated DNA fragments were column purified and eluted in 20 µL of elution buffer and stored at − 20 °C till further used for library preparation.
5
Methylation Detection via MeDIP Assay
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Methylation was detected by the MeDIP kit (Abcam, London, United Kingdom) by applying a non-cross-reactive 5mC antibody. In the assay of the kit, DNA is sheared and then added into a microplate well and an antibody specific to methylcytosine is then used to capture methylated DNA in the wells. Then, the specificity of enriched methylated DNA was evaluated by quantitative PCR.
6
Methylation Analysis of LINE-1 Promoter
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LINE-1 5=-UTR promoter methylation analysis was performed on isolated gastrocnemius DNA (described above) from n ϭ 8 rats per age group using a commercially available methylated DNA immunoprecipitation (MeDIP) kit (product no. ab117133; Abcam). Before the assay as performed, 1.5 g of gastrocnemius DNA was digested using MseI (New England BioLabs, Ipswich, MA). Following digestion reactions, total methylated DNA from a total of 1 g input was immunoprecipitated using an anti-5-methylcytosine antibody provided within the kit. RT-PCR was then performed on 25 ng of the methylated DNA using the L1.3 primers described above to decipher fold change in methylated LINE-1 5=-UTR. Residual input DNA from each sample (25 ng) was used as a control to normalize RT-PCR results. Fold change in 5-UTR promoter methylation was calculated using the 2 ⌬⌬Cq method where 2 ⌬Cq ϭ 2^[input L1.3 DNA Cqmethylated L1.3 DNA Cq] and 2 ⌬⌬Cq (or fold change) ϭ [2 ⌬Cq value/2 ⌬Cq average of 3 mo age group]. Fold change values from 3-mo-old rats were performed using the aforementioned 2 ⌬⌬Cq method, and data are presented as REUs. Overall coefficient of variation values for Cq triplicates of the assayed genes were as follows: input L1.3 ϭ 0.44% and methylated L1.3 ϭ 0.35%.
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