The Methyl Primer Express v1.0 software (Applied Biosystems, Life technologies, Grand Island, New York) was used to identify the CpG islands in gene promoter regions and to design specific primers for the methylation analysis (S1 Table ). DNA methylation status was established by bisulfite genomic sequencing. DNA was extracted from samples using the DNeasy tissue kit (Qiagen, Milan, Italy) and 1 μg of DNA was modified with sodium bisulfite using the EZ DNA methylation-gold kit (Zymo Research, CA USA) according to manufacturer´s instructions. Multiple clones were analyzed for each sample and the methylation frequency was calculated in each case.
Methyl primer express v1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Methyl Primer Express v1.0 is a software tool designed for the design of bisulfite-converted DNA primer sequences. It facilitates the identification of optimal primer sets for the analysis of DNA methylation patterns.
Automatically generated - may contain errors
Lab products found in correlation
Ez dna methylation gold kit, by Zymo Research (7 mentions)
Pgem t easy vector, by Promega (5 mentions)
Epitect bisulfite kit, by Qiagen (5 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (4 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (4 mentions)
Nucleospin 96 plasmid, by Macherey-Nagel (3 mentions)
Immolase taq polymerase, by Meridian Bioscience (3 mentions)
Pgem t easy vector system, by Promega (3 mentions)
Ez dna methylation kit, by Zymo Research (3 mentions)
Epitect methyl qpcr assay, by Qiagen (2 mentions)
Wizard sv gel and pcr clean up system, by Promega (2 mentions)
Amplitaq gold, by Thermo Fisher Scientific (2 mentions)
Dp215, by Tiangen Biotech (2 mentions)
Em101, by Tiangen Biotech (2 mentions)
Kapa sybr fast qpcr kit, by Roche (2 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Hrm master mix, by Qiagen (1 mentions)
Precision melt analysis software, by Bio-Rad (1 mentions)
Cfx96 real time pcr machine, by Bio-Rad (1 mentions)
Wizard plus sv minipreps dna purification system, by Promega (1 mentions)
41 protocols using methyl primer express v1
1
Bisulfite Genomic Sequencing for DNA Methylation
2
High-Sensitivity Epigenetic Methylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
High resolution melting PCR enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology, which is widely used to measure relative DNA methylation levels in pre-clinical or clinical epigenetic studies (Wojdacz et al., 2008 (link)). HRM-PCR was performed on bisulfite-converted DNA using primer sequences that were designed to indiscriminately amplify both methylated and unmethylated bisulfite-converted DNA. The bisulfite-specific PCR primers (Supplementary Table S2 ) targeting two regions of the CpG island within the proximal promoter region of the SLC1A2 gene were designed using MethylPrimer Express v1.0 software (Applied Biosystems, Foster City, CA, United States).
Each reaction was carried out using 1 ng/μL bisulfite-converted DNA, HRM Master Mix (Qiagen), and 0.5 μM of each primer in a total reaction volume of 10 μL. HRM-PCR was performed on a CFX96 Real-Time PCR machine (Bio-Rad, Hercules, CA, United States) in triplicate. Enzyme activation was carried out for 5 min at 95°C and followed by 42 cycles of the following steps: 10 s of denaturation at 95°C, 30 s of annealing at 58°C, and 24 s of extension at 72°C. Samples were then warmed to 95°C at +0.1°C per second. Precision Melt AnalysisTM software (Bio-Rad, Hercules, CA, United States) was used to analyze PCR product melting temperatures.
Each reaction was carried out using 1 ng/μL bisulfite-converted DNA, HRM Master Mix (Qiagen), and 0.5 μM of each primer in a total reaction volume of 10 μL. HRM-PCR was performed on a CFX96 Real-Time PCR machine (Bio-Rad, Hercules, CA, United States) in triplicate. Enzyme activation was carried out for 5 min at 95°C and followed by 42 cycles of the following steps: 10 s of denaturation at 95°C, 30 s of annealing at 58°C, and 24 s of extension at 72°C. Samples were then warmed to 95°C at +0.1°C per second. Precision Melt AnalysisTM software (Bio-Rad, Hercules, CA, United States) was used to analyze PCR product melting temperatures.
3
Methylation Analysis of LINC00473 Promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methyl Primer Express v1.0 software (Applied Biosystems) and Primer3 (v.0.4.0) were used to design primers for the methylation analysis. DNA (1 µg) was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Gold kit (Zymo Research). A 490-bp fragment of the LINC00473 promoter was amplified using 2 μL of bisulfite-converted DNA with ImmolaseTaq polymerase (Bioline) at 60 °C for 40 cycles. The resulting PCR product was gel-purified (2% agarose) with NucleoSpin® Gel and PCR Clean-up (Macherey–Nagel) and then cloned into the pGEMT Easy Vector System (Promega) following the manufacturers’ protocols. For all samples, 10 colonies were randomly chosen, and DNA was purified using NucleoSpin® 96 Plasmid (Macherey–Nagel) and sequenced with a 3730 DNA Analyzer (Applied Biosystems). Results were transformed into percentages of CpGs showing methylation.
4
CpG Island Analysis and Primer Design for PTCH1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CpG island analysis and primer design for PTCH1 mRNA of the transcription start site (counted as 0:00) −3950 bp upstream and +2050 bp downstream were conducted with Methyl Primer Express® v1.0 software (Applied Biosystems; Life Technologies). Table I lists the MSP primers. PCR cooling amplification was performed in 8 μl bisulfite-treated DNA with a conventionally configured reaction system. Briefly, pre-degeneration was completed at 95°C for 5 min. Prior to PCR, predegeneration was completed at 95°C for 5 min. Next, cooling PCR was performed for 10 cycles (PCR degeneration at 94°C for 30 sec, with renaturation temperature decreases of 0.5°C per cycle; temperature decreases from +3°C to −2°C for 30 sec; and extension at 72°C for 30 sec). Next, the normal PCR was performed for 40 cycles (PCR degeneration at 94°C for 30 sec; −2°C renaturation for 30 sec; and extension at 72°C for 30 sec). Amplification products were analyzed by 1.5% agarose gel electrophoresis.
5
Methylation Analysis of HMGA2/RPSAP52 Island
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Methyl Primer Express v1.0 software (Applied Biosystems) was used to design specific primers for the methylation analysis of HMGA2/RPSAP52 island (Supplementary Table 1 ). Genomic DNA (1 µg) was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Gold kit (Zymo Research). For bisulfite genomic sequencing, 300–500 bp fragments were amplified using 1–2 μl of bisulfite-converted DNA with Immolase Taq polymerase (Bioline) for 42 cycles. The resulting PCR products were gel-purified with NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel) and then cloned into the pSpark® TA vector (Canvas) according to the manufacturer’s protocol. For all samples, 10 colonies were randomly chosen, the DNA was purified using NucleoSpin® 96 Plasmid (Macherey-Nagel) and sequenced by the 3730 DNA Analyzer (Applied Biosystems). After sequencing analysis with BioEdit v7.2.5 software, C nucleotides that remained unaltered were transformed into percentages of CpGs showing methylation.
6
Quantifying DNA Methylation Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methylation-specific real-time qPCR primers (Table 1 ) for CpG-rich regions were designed using Methyl Primer Express v1.0 software (Applied Biosystems, CA, USA). Quantification of DNA methylation status was determined using the EpiTect Methyl qPCR assay (SABiosciences, Frederick, MD) by following the manufacturer’s protocol. Briefly, gDNA was digested with a combination of methylation-sensitive, methylation-dependent, and both methylation-sensitive and methylation-dependent enzymes or without enzyme added (mock) at 37 °C for 16 h. After enzyme inactivation at 65 °C for 20 min, real-time qPCR was carried out according to the EpiTect protocol. All reactions were performed in triplicate. Relative fractions of methylated and unmethylated DNA were measured by comparing the amount in each digest with that of the mock digest using the ΔCt method.
7
Methylation Analysis of CDKN1C Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methyl Primer Express v1.0 software (Applied Biosystems) was used to identify CpG islands around the Transcriptional Star Site (TSS) of CDKN1C gene, and to design specific primers for the methylation analysis. DNA (1 μg) was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Gold kit (Zymo Research, CA, USA). MSP was performed with primers specific for methylated (M) or unmethylated (U) CpG sites. For bisulfite genomic sequencing, a region included in the one analysed by MSP was amplified using 1 μL of bisulfite-converted DNA with Immolase Taq polymerase (Bioline USA Inc., Kenilworth, NJ) at 60 °C for 40 cycles. Then the resulting PCR products were gel-purified (2% agarose) with Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and cloned into the pGEMT Easy Vector System (Promega) following the manufacturer-specific protocols. For all samples, 12 colonies were randomly chosen, and DNA was purified using Wizard® Plus SV Minipreps DNA Purification System (Promega) and sequenced with a ABI 3730 xl DNA Analyzer (Applied Biosystems). After sequencing analysis, the results were transformed into percentages of CpGs calculated in comparison with the total CpGs of the analysed region. Primers and conditions are indicated in Additional file 4 : Table S2.
8
CaSR Promoter CpG Island Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify the CpG island/s in the CaSR promoter region we used the following settings of Methyl Primer Express v1.0 Software (Applied Biosystems, Austria): minimum length 300 bp, maximum length 2,000 bp, C+Gs/total bases >50%, and CpGs observed/CpGs expected >0.6. The same software was used to design primers for bisulfite-specific PCR. Detailed description of primer sequences and PCR conditions are provided in Supporting Information Table 1.
DNA was isolated by digestion with proteinase K following phenol/chloroform extraction. DNA was bisulfite modified using EpiTect Bisulfite Kit. PCR amplification was performed with HotStarTaq DNA Polymerase. PCR products were run on a 2% low-melt agarose gel, bands were excised, and purified with PureLink Quick Gel Extraction Kit. Cloning was performed with the Topo TA Cloning Kit, electro-competent bacteria were used for subcloning following the recommended protocol. Bacterial cultures were grown in lysogeny broth media containing 50 µg ml−1 kanamycin. Minipreps were performed with PureLink Quick Plasmid Miniprep Kit and DNA sequencing was performed in Genetic Analyzer 3130xI (Applied Biosystems). For each sample multiple clones were sequenced. Results were analyzed using BiQ Analyzer software.34 (link) Pyrosequencing of the CaSR promoter was carried out by EpigenDX, MA.
DNA was isolated by digestion with proteinase K following phenol/chloroform extraction. DNA was bisulfite modified using EpiTect Bisulfite Kit. PCR amplification was performed with HotStarTaq DNA Polymerase. PCR products were run on a 2% low-melt agarose gel, bands were excised, and purified with PureLink Quick Gel Extraction Kit. Cloning was performed with the Topo TA Cloning Kit, electro-competent bacteria were used for subcloning following the recommended protocol. Bacterial cultures were grown in lysogeny broth media containing 50 µg ml−1 kanamycin. Minipreps were performed with PureLink Quick Plasmid Miniprep Kit and DNA sequencing was performed in Genetic Analyzer 3130xI (Applied Biosystems). For each sample multiple clones were sequenced. Results were analyzed using BiQ Analyzer software.34 (link) Pyrosequencing of the CaSR promoter was carried out by EpigenDX, MA.
9
TMPRSS4 Promoter Methylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Methyl Primer Express v1.0 software (Applied Biosystems) was used to identify the TMPRSS4 promoter CpGs and to design specific primers for the methylation analysis (Supplementary Table 4 ). None of the primers covered any CpG. Approximately 1 μg of DNA from each human lung cancer cell line was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Gold kit (Zymo Research). A 394-bp fragment, −199 bp to +195 bp relative to the transcription start site (TSS), was amplified using 2 μL of bisulfite-converted DNA with Immolase Taq polymerase (Bioline) at 60°C for 35-40 cycles. The resulting PCR products were gel-purified (2% agarose) with NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel) and then cloned into the pGEMT Easy Vector System (Promega) according to the manufacturer's protocol. In all the samples, 10 colonies were randomly chosen, and DNA was purified using NucleoSpin® 96 Plasmid (Macherey-Nagel) and sequenced. After sequencing analysis, results were transformed into percentages of CpGs showing methylation.
10
Quantitative SYBR Green Methylation-Specific PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative SYBR green methylation-specific PCR (QSG-MSP) QSG-MSP was performed to quantify the levels of CpG DNA methylation of SKI using the Applied Biosystems 7500 real time PCR system (Applied Biosystems, Foster City, CA) as previously reported [12] . Primers for QSG-MSP were designed using Methyl Primer Express Software v1.0 (Applied Biosystems). The presence of CpG islands was determined using Methyl Primer Express v1.0 software (Applied Biosystems). Primer sequences were 5′-TAAAGTCGGGGATGGTAGGAC-3′ (forward) and 5′-CGATCGCGATTCTTAAAAAAC-3′ (reverse). Primers for the control ACTB (beta-actin) gene were 5′-TGGTGATGGAGGAGG TTTAGTAAGT-3′ (forward) and 5′-AACCAATAAAACCTACTCCTCCCT TAA-3′ (reverse) as previously described [12] . Quantitative PCR was performed in a 25-μL reaction volume with 12.5 μL of 2 × SYBR Green PCR Master Mix (Applied Biosystems), 2.5 pmol of each primer, and 25 ng of bisulfite-treated DNA sample. Thermal cycling was as follows: 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Only samples with amplification at the correct melting temperature were used for further analyses of methylation. The amount of methylated DNA (percentage of methylated reference) was calculated as follows: ratio of quantity of target gene to quantity of target gene of test sample divided by quantity of ACTB.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!