What precautions and/or quality controls should I use when preparing my samples to ensure a good experiment?
Do you recommend duplicates or triplicates when running your assays?
Do you use sonication to fragment the DNA, and what are the sizes of the resulting fragments?
What procedures do you use to qualify antibodies for ChIP applications?
What if there are no antibodies available for my transcription factor?
Do you recommend using IgG as a control antibody?
What are your sample requirements for ChIP Sequencing or ChIP-on-Chip assays?
In the case of adherent cells, do you want the cultured cells attached or trypsinized and sent in tubes
What controls should we include for a Query assay?
What if we are not sure how much tissue will be needed?
What is the difference between the Affymetrix Tiling Array Software (TAS) and the Model-Based Analysis of Tiling-Array (MAT) software developed by Shirley Liu’s laboratory? What are the benefits of each?
How long does it take to carry out a ChIP Sequencing or ChIP-on-Chip assay?
What are the benefits of using the whole-genome tiling array set versus a promoter array for a ChIP-on-ChIP study?
What are the advantages of a ChIP Sequencing assay versus a ChIP-on-ChIP assay?
Can you give us an overview of the procedural steps that our samples will undergo during a ChIP/DNA IP assay at Genpathway?
How much antibody is needed for an antibody test and/or assay?
How much sample is used for the antibody test?
What amplification method do you use?
Why do I need to purchase an additional Affymetrix array set for Input DNA?
For a typical ChIP Sequencing run, what is the sequence tag length and how does that typically relate to target binding site coverage? Do you achieve at least 10X coverage?
What data results will Genpathway provide?
Are transcription factor binding results from the Illumina ChIP Sequencing output quantitative or qualitative?
Our IT department is overwhelmed with the prospect of the enormous amounts of data per run. Will handling of data on that scale be problematic?
What material should I send for the DNA methylation assay?
Do you have a preference between DNA or cells/tissue for the DNA methylation assay?
How do Genpathway’s ChIP/DNA IP protocols compare with commercially available ChIP kits?
What precautions and/or quality controls should I use when preparing my samples to ensure a good experiment?
From a technical standpoint, Genpathway has shown excellent reproducibility in all of its ChIP/DNA IP assays and suggests that replicates are not required. From the biological standpoint, each investigator will need to assess the need for replicates in his/her biological system.
We qualify antibodies by using one of the two following ChIP assays:
Query (QPCR): If binding sites are known (or proposed) for the factor of interest, small ChIP reactions using your samples are carried out to assess both absolute and relative signals at the known/proposed positive sites compared to negative genomic regions.
ChIP-on-Chip: If no binding sites are known, a ChIP reaction using an appropriate chromatin sample is carried out followed by amplification and hybridization to a tiling array. The number and heights of the peaks are assessed to determine if the antibody works in ChIP.
What if there are no antibodies available for my transcription factor?
You can consider expressing a tagged version of your transcription factor in cells. Many antibodies that recognize peptide tags work well in ChIP. Genpathway has carried out ChIP with antibodies that recognize the Flag, T7, V5, HA and Myc tags.
Do you recommend using IgG as a control antibody?
In general, we recommend using controls other than IgG. Control IgG’s typically show lower background binding than antibodies against specific factors. For Query (QPCR) assays, we carry out QPCR with primers that target genomic regions not expected to be bound by the factor being studied. We have found that this control is usually more stringent than an IgG control.
For ChIP-on-ChIP, one control needed is a parallel hybridization using DNA from across the genome. This is necessary to normalize the hybridization signal at each probe. For this, we recommend using unprecipitated genomic DNA (Input DNA) rather than control IgG ChIP DNA because some genomic sequences may be overrepresented and some may be underrepresented in the IgG IP.
For ChIP Sequencing, again we recommend using Input DNA rather than control IgG ChIP DNA when controlling for local copy number changes in the sample genome.
What are your sample requirements for ChIP Sequencing or ChIP-on-Chip assays?
The amounts vary depending on the types of samples and the assay parameters. Genpathway will be happy to discuss the requirements with you. Cell numbers required typically range from 1-10 million and tissue amounts are generally in the 50-500 mg range per ChIP reaction.
In the case of adherent cells, do you want the cultured cells attached or trypsinized and sent in tubes?
Cells that grow attached to flask or plate surfaces (adherent cells) should be fixed and transferred to tubes according to Genpathway’s fixation protocol. For this, the formaldehyde is added directly to the media of the cultures without use of trypsin. After termination of fixation using the glycine step, a cell scraper is used to scrape the cells from the surface of the flasks or plates, and the cells are collected into tube(s). The remainder of the fixation protocol should then be followed.
If adherent cells have been trypsinized, collected in tubes and frozen without prior fixation (with liquid decanted before freezing), they can be sent as is to Genpathway for processing into chromatin or DNA.
What controls should we include for a Query assay?
The customer need not provide “ChIP controls”. For Query (QPCR), you should provide Genpathway with genomic regions expected to be bound by the factor of interest.
If cell controls are desired, they should be designed according to the biological system being assayed, e.g., untreated or vehicle-treated cells vs. treatment with a compound.
What if we are not sure how much tissue will be needed?
Contact Genpathway and we will advise you based on our prior experience with different tissues. Genpathway can also carry out a chromatin yield test on representative tissue in order for you to know how much to send for your assay.
What is the difference between the Affymetrix Tiling Array Software (TAS) and the Model-Based Analysis of Tiling-Array (MAT) software developed by Shirley Liu’s laboratory? What are the benefits of each?
TAS is the Tiling Array Software developed by Affymetrix for analysis of their tiling arrays. It compares the signals from each test ChIP array with the signals from the control array (usually hybridized with Input DNA), expresses the comparison for each probe as a ratio, and then averages those ratios over a sliding window in order to take into account nearest neighbor probes. MAT was developed by Dr. Liu’s group as an independent approach for analyzing tiling array data. Genpathway has compared results generated by the two software programs on the same array data and found them to be very similar.
How long does it take to carry out a ChIP Sequencing or ChIP-on-Chip assay?
A ChIP Sequencing assay takes about 8-10 weeks from the time we receive your samples. A ChIP-on-Chip assay takes about 5-6 weeks.
What are the benefits of using the whole-genome tiling array set versus a promoter array for a ChIP-on-ChIP study?
The Affymetrix promoter arrays for human and mouse are tiled across over 25,000 promoters each. Each promoter region consists of approximately 7.5 kb upstream and 2.5 kb downstream of the transcription start site (TSS). Therefore, while protein-associated sites and methylated DNA regions within those regions will be identified, clearly the number of regions being examined is limited when using promoter arrays. Over the past 5 years, we (and others) have found that binding sites for transcription factors, histone marks and sites of DNA methylation are not restricted to promoters and in fact are generally found within genes, far upstream of genes and downstream of genes. For some transcription factors, e.g., steroid hormone receptors, only 2-10% of binding occurs in promoters.
What are the advantages of a ChIP Sequencing assay versus a ChIP-on-ChIP assay?
Both types of assays, when carried out by Genpathway, are high-quality whole-genome assays. Advantages of ChIP Sequencing include a moderate increase in genome coverage due to repeat-masking of the arrays, increased resolution of the binding sites, the ability to carry out assays in any genome for which at least some genomic sequence and annotation is known vs. only human and mouse tiling arrays sets for ChIP-on-Chip, and a lower cost.
One advantage for ChIP-on-Chip includes the option to carry out a scaled down version using promoter arrays. Also, some antibodies may enrich for sites present in highly repetitive regions such as centromeres, e.g., methylated DNA regions. ChIP Sequencing of this ChIP DNA may result in many tags that do not map uniquely and that dilute out sequencing of more informative regions. For these types of assays, ChIP-on-Chip may be better because repetitive regions are not represented on the arrays.
Can you give us an overview of the procedural steps that our samples will undergo during a ChIP/DNA IP assay at Genpathway?
The process will vary slightly depending on the samples sent and assay being performed. In general, the steps are:
If the customer is providing the antibody, typically 4 micrograms of antibody are required per ChIP reaction.
How much sample is used for the antibody test?
10-20 micrograms of chromatin (measurement refers to the DNA component of chromatin).
What amplification method do you use?
For ChIP Sequencing, we use Illumina’s protocol for amplification, which is also referred to as “library generation”. For ChIP-on-Chip or MeDNA IP-on-Chip, we use the whole genome amplification (WGA) kit from Sigma.
Why do I need to purchase an additional Affymetrix array set for Input DNA?
Affymetrix arrays are tiled with 25-mer probes across the genome (except for repetitive sequences). These probes exhibit different hybridization kinetics due to varying GC-content and sequence composition. Therefore, hybridization with Input DNA is necessary to normalize for differences in hybridization efficiencies of the probes. Additionally, the control array normalizes for copy number variations that could otherwise result in false positives. Affymetrix tiling arrays (tiled at very high density) were developed such that the control hybridizations for each array type are carried out on a separate array. Thus, one additional set of Input DNA arrays when using the 7-array full genome set (or one array if using only one array type or the promoter array) is required in each assay.
For a typical ChIP Sequencing run, what is the sequence tag length and how does that typically relate to target binding site coverage? Do you achieve at least 10X coverage?
Using Illumina sequencing, the sequence tags are typically 35 bp in length. The amount of sequence requested is 250 megabases (MB), which corresponds to 7-8 million sequences [need to check our numbers]. This amount of sequencing is now attainable using one channel in a GAII flow cell. Typically 80% of “quality-filtered” sequences (those that pass the quality criteria) will map uniquely, and the coverage at each site depends on the binding strength for the factor at that site. Coverage at a given binding site can be as many as 200 tags and most sites have at least 10X coverage (this depends on the number of tags sequenced).
What data results will Genpathway provide?
For ChIP Query, you will receive an excel file containing QPCR results averaged from triplicate reactions for the genomic regions/genes of interest and negative control regions. Each file includes the raw data, normalized/calculated results, and a graph.
For ChIP-on-ChIP and ChIP Sequencing, the following files are provided (smaller files by email and the remainder on a CD/DVD):
Measurements of TF binding should be considered semi-quantitative due to amplification of the ChIP DNAs prior to sequencing. Genpathway makes every effort to carry out the amplification in as linear a fashion as possible, although some bias in amplification efficiencies may occur between different types of sequences. When differential binding levels have been validated using Genpathway’s Query assay, the differential binding observed between samples or between sites agreed very well with the ChIP Sequencing numbers, demonstrating good quantitation using this type of whole-genome assay.
Our IT department is overwhelmed with the prospect of the enormous amounts of data per run. Will handling of data on that scale be problematic?
Genpathway’s comprehensive analysis of ChIP Sequencing data condenses the results into manageable files that can be directly data-mined. While the raw data files are always sent to the customer as well, there is no need for the customer to deal with the large raw data files. In addition, Genpathway scientists are available for consultation regarding more complex analyses as desired.
What material should I send for the DNA methylation assay?
You can send purified DNA or frozen cells or tissues. If purified DNAs are sent, we ask that they be treated with RNase before the final concentrations are determined, and that 200-400 ng of each DNA be run on a 1% agarose gel to verify the DNA is of high molecular weight.
If cells or tissues are sent, they should not be fixed. Simply freeze the tissue or cell pellets and Genpathway will extract the DNA.
Do you have a preference between DNA or cells/tissue for the DNA methylation assay?
This is up to the customer, but if there is a choice, we prefer that cells or tissue are sent so Genpathway can extract the DNA. If DNA is sent, Genpathway still goes through a clean-up process with RNase and DNA purification followed by a final determination of DNA concentration. Results from this additional processing are also sent to the customer.
How do Genpathway’s ChIP/DNA IP protocols compare with commercially available ChIP kits?
Genpathway has optimized its protocols for better sensitivity and specificity (the latter referring to pull-down of significantly less background DNA). Recently, a direct comparison of Genpathway’s protocols with 5 ChIP kits showed that Genpathway’s protocols performed in a significantly superior manner. You may request more information about the comparison from Genpathway.
Do you recommend duplicates or triplicates when running your assays?
Do you use sonication to fragment the DNA, and what are the sizes of the resulting fragments?
What procedures do you use to qualify antibodies for ChIP applications?
What if there are no antibodies available for my transcription factor?
Do you recommend using IgG as a control antibody?
What are your sample requirements for ChIP Sequencing or ChIP-on-Chip assays?
In the case of adherent cells, do you want the cultured cells attached or trypsinized and sent in tubes
What controls should we include for a Query assay?
What if we are not sure how much tissue will be needed?
What is the difference between the Affymetrix Tiling Array Software (TAS) and the Model-Based Analysis of Tiling-Array (MAT) software developed by Shirley Liu’s laboratory? What are the benefits of each?
How long does it take to carry out a ChIP Sequencing or ChIP-on-Chip assay?
What are the benefits of using the whole-genome tiling array set versus a promoter array for a ChIP-on-ChIP study?
What are the advantages of a ChIP Sequencing assay versus a ChIP-on-ChIP assay?
Can you give us an overview of the procedural steps that our samples will undergo during a ChIP/DNA IP assay at Genpathway?
How much antibody is needed for an antibody test and/or assay?
How much sample is used for the antibody test?
What amplification method do you use?
Why do I need to purchase an additional Affymetrix array set for Input DNA?
For a typical ChIP Sequencing run, what is the sequence tag length and how does that typically relate to target binding site coverage? Do you achieve at least 10X coverage?
What data results will Genpathway provide?
Are transcription factor binding results from the Illumina ChIP Sequencing output quantitative or qualitative?
Our IT department is overwhelmed with the prospect of the enormous amounts of data per run. Will handling of data on that scale be problematic?
What material should I send for the DNA methylation assay?
Do you have a preference between DNA or cells/tissue for the DNA methylation assay?
How do Genpathway’s ChIP/DNA IP protocols compare with commercially available ChIP kits?
What precautions and/or quality controls should I use when preparing my samples to ensure a good experiment?
- When preparing cells, carry out your treatments or culturing conditions and then follow the Genpathway fixation procedure exactly as written.
- When sending tissues, freeze the tissues on dry ice as soon as possible after removal and store at -70 C before shipment.
-
When sending DNA for methylation analysis, it should be treated with RNase prior to determining the final DNA concentration. Run 200-400 ng of each DNA on a gel to verify the DNA is of high molecular weight and not degraded.
Do you use sonication to fragment the DNA, and what are the sizes of the resulting fragments?
Yes, we shear the DNA by sonication. The resulting DNA fragments are predominantly 300-500 bp.
What procedures do you use to qualify antibodies for ChIP applications?Yes, we shear the DNA by sonication. The resulting DNA fragments are predominantly 300-500 bp.
We qualify antibodies by using one of the two following ChIP assays:
Query (QPCR): If binding sites are known (or proposed) for the factor of interest, small ChIP reactions using your samples are carried out to assess both absolute and relative signals at the known/proposed positive sites compared to negative genomic regions.
ChIP-on-Chip: If no binding sites are known, a ChIP reaction using an appropriate chromatin sample is carried out followed by amplification and hybridization to a tiling array. The number and heights of the peaks are assessed to determine if the antibody works in ChIP.
What if there are no antibodies available for my transcription factor?
You can consider expressing a tagged version of your transcription factor in cells. Many antibodies that recognize peptide tags work well in ChIP. Genpathway has carried out ChIP with antibodies that recognize the Flag, T7, V5, HA and Myc tags.
Do you recommend using IgG as a control antibody?
In general, we recommend using controls other than IgG. Control IgG’s typically show lower background binding than antibodies against specific factors. For Query (QPCR) assays, we carry out QPCR with primers that target genomic regions not expected to be bound by the factor being studied. We have found that this control is usually more stringent than an IgG control.
For ChIP-on-ChIP, one control needed is a parallel hybridization using DNA from across the genome. This is necessary to normalize the hybridization signal at each probe. For this, we recommend using unprecipitated genomic DNA (Input DNA) rather than control IgG ChIP DNA because some genomic sequences may be overrepresented and some may be underrepresented in the IgG IP.
For ChIP Sequencing, again we recommend using Input DNA rather than control IgG ChIP DNA when controlling for local copy number changes in the sample genome.
What are your sample requirements for ChIP Sequencing or ChIP-on-Chip assays?
The amounts vary depending on the types of samples and the assay parameters. Genpathway will be happy to discuss the requirements with you. Cell numbers required typically range from 1-10 million and tissue amounts are generally in the 50-500 mg range per ChIP reaction.
In the case of adherent cells, do you want the cultured cells attached or trypsinized and sent in tubes?
Cells that grow attached to flask or plate surfaces (adherent cells) should be fixed and transferred to tubes according to Genpathway’s fixation protocol. For this, the formaldehyde is added directly to the media of the cultures without use of trypsin. After termination of fixation using the glycine step, a cell scraper is used to scrape the cells from the surface of the flasks or plates, and the cells are collected into tube(s). The remainder of the fixation protocol should then be followed.
If adherent cells have been trypsinized, collected in tubes and frozen without prior fixation (with liquid decanted before freezing), they can be sent as is to Genpathway for processing into chromatin or DNA.
What controls should we include for a Query assay?
The customer need not provide “ChIP controls”. For Query (QPCR), you should provide Genpathway with genomic regions expected to be bound by the factor of interest.
If cell controls are desired, they should be designed according to the biological system being assayed, e.g., untreated or vehicle-treated cells vs. treatment with a compound.
What if we are not sure how much tissue will be needed?
Contact Genpathway and we will advise you based on our prior experience with different tissues. Genpathway can also carry out a chromatin yield test on representative tissue in order for you to know how much to send for your assay.
What is the difference between the Affymetrix Tiling Array Software (TAS) and the Model-Based Analysis of Tiling-Array (MAT) software developed by Shirley Liu’s laboratory? What are the benefits of each?
TAS is the Tiling Array Software developed by Affymetrix for analysis of their tiling arrays. It compares the signals from each test ChIP array with the signals from the control array (usually hybridized with Input DNA), expresses the comparison for each probe as a ratio, and then averages those ratios over a sliding window in order to take into account nearest neighbor probes. MAT was developed by Dr. Liu’s group as an independent approach for analyzing tiling array data. Genpathway has compared results generated by the two software programs on the same array data and found them to be very similar.
How long does it take to carry out a ChIP Sequencing or ChIP-on-Chip assay?
A ChIP Sequencing assay takes about 8-10 weeks from the time we receive your samples. A ChIP-on-Chip assay takes about 5-6 weeks.
What are the benefits of using the whole-genome tiling array set versus a promoter array for a ChIP-on-ChIP study?
The Affymetrix promoter arrays for human and mouse are tiled across over 25,000 promoters each. Each promoter region consists of approximately 7.5 kb upstream and 2.5 kb downstream of the transcription start site (TSS). Therefore, while protein-associated sites and methylated DNA regions within those regions will be identified, clearly the number of regions being examined is limited when using promoter arrays. Over the past 5 years, we (and others) have found that binding sites for transcription factors, histone marks and sites of DNA methylation are not restricted to promoters and in fact are generally found within genes, far upstream of genes and downstream of genes. For some transcription factors, e.g., steroid hormone receptors, only 2-10% of binding occurs in promoters.
What are the advantages of a ChIP Sequencing assay versus a ChIP-on-ChIP assay?
Both types of assays, when carried out by Genpathway, are high-quality whole-genome assays. Advantages of ChIP Sequencing include a moderate increase in genome coverage due to repeat-masking of the arrays, increased resolution of the binding sites, the ability to carry out assays in any genome for which at least some genomic sequence and annotation is known vs. only human and mouse tiling arrays sets for ChIP-on-Chip, and a lower cost.
One advantage for ChIP-on-Chip includes the option to carry out a scaled down version using promoter arrays. Also, some antibodies may enrich for sites present in highly repetitive regions such as centromeres, e.g., methylated DNA regions. ChIP Sequencing of this ChIP DNA may result in many tags that do not map uniquely and that dilute out sequencing of more informative regions. For these types of assays, ChIP-on-Chip may be better because repetitive regions are not represented on the arrays.
Can you give us an overview of the procedural steps that our samples will undergo during a ChIP/DNA IP assay at Genpathway?
The process will vary slightly depending on the samples sent and assay being performed. In general, the steps are:
- Samples are processed to isolate the chromatin or DNA (also includes sonication). The resulting chromatin or DNA isolates are quantified and the yields are sent to the customer.
- Antibody tests (using Genpathway’s Query (QPCR) assay or hybridization to a tiling array) are performed as needed using one or several of the customer samples. Results are sent to the customer and additional antibodies are tested as needed.
- ChIP/DNA IP reactions are carried out with the chromatin or DNA, followed by reversal of cross-links (for chromatin) and isolation of the resulting enriched ChIP/IP DNA.
- ChIP/DNA IP reactions are QC’ed using Genpathway’s Query (QPCR) assay to measure signals at positive regions expected to be bound by the factor in comparison to negative control regions.
- If the samples are to be tested by ChIP Sequencing or ChIP-on-Chip, the ChIP/IP DNAs are amplified using appropriate protocols.
- Amplification reactions are QC’ed using Genpathway’s Query (QPCR) assay to ensure that binding sites enriched in the post-ChIP/DNA IP Query are present with good enrichment post-amplification.
- The amplified ChIP/IP DNAs are sequenced or labeled and hybridized to arrays (for ChIP Sequencing or ChIP-on-Chip, respectively).
- Data are comprehensively analyzed, and the results are sent to the customer.
If the customer is providing the antibody, typically 4 micrograms of antibody are required per ChIP reaction.
How much sample is used for the antibody test?
10-20 micrograms of chromatin (measurement refers to the DNA component of chromatin).
What amplification method do you use?
For ChIP Sequencing, we use Illumina’s protocol for amplification, which is also referred to as “library generation”. For ChIP-on-Chip or MeDNA IP-on-Chip, we use the whole genome amplification (WGA) kit from Sigma.
Why do I need to purchase an additional Affymetrix array set for Input DNA?
Affymetrix arrays are tiled with 25-mer probes across the genome (except for repetitive sequences). These probes exhibit different hybridization kinetics due to varying GC-content and sequence composition. Therefore, hybridization with Input DNA is necessary to normalize for differences in hybridization efficiencies of the probes. Additionally, the control array normalizes for copy number variations that could otherwise result in false positives. Affymetrix tiling arrays (tiled at very high density) were developed such that the control hybridizations for each array type are carried out on a separate array. Thus, one additional set of Input DNA arrays when using the 7-array full genome set (or one array if using only one array type or the promoter array) is required in each assay.
For a typical ChIP Sequencing run, what is the sequence tag length and how does that typically relate to target binding site coverage? Do you achieve at least 10X coverage?
Using Illumina sequencing, the sequence tags are typically 35 bp in length. The amount of sequence requested is 250 megabases (MB), which corresponds to 7-8 million sequences [need to check our numbers]. This amount of sequencing is now attainable using one channel in a GAII flow cell. Typically 80% of “quality-filtered” sequences (those that pass the quality criteria) will map uniquely, and the coverage at each site depends on the binding strength for the factor at that site. Coverage at a given binding site can be as many as 200 tags and most sites have at least 10X coverage (this depends on the number of tags sequenced).
What data results will Genpathway provide?
For ChIP Query, you will receive an excel file containing QPCR results averaged from triplicate reactions for the genomic regions/genes of interest and negative control regions. Each file includes the raw data, normalized/calculated results, and a graph.
For ChIP-on-ChIP and ChIP Sequencing, the following files are provided (smaller files by email and the remainder on a CD/DVD):
- Excel data files.
a. Intervals. Information on all peaks, including their genomic locations, metrics for quantitation, and relationships to genes and other genomic annotations.
b. Genes. Information on all genes associated with the peaks. Also includes some of the Interval information plus additional information about the genes.
c. Active Regions. Information on all genomic regions that contain peaks, including their locations, Interval metrics, relationships to genes, and comparisons across samples. - Excel summary file. Correlations across samples in the numbers of Intervals and Active Regions, other assay statistics, and analysis parameters.
- Interval sequences. DNA sequences of the Intervals.
- BED file. File for uploading as a custom track into the UCSC browser for visualization of Intervals, Active Regions and Interval peaks.
- Raw data sequence aligns for ChIP Sequencing; raw data CEL files for ChIP-on-Chip.
- Signal BAR files for uploading into the Integrated Genome Browser (downloadable from Affymetrix) for visualization of all probe signals and peaks.
- Data explanation document. Explanation of the analysis process and data files provided.
Measurements of TF binding should be considered semi-quantitative due to amplification of the ChIP DNAs prior to sequencing. Genpathway makes every effort to carry out the amplification in as linear a fashion as possible, although some bias in amplification efficiencies may occur between different types of sequences. When differential binding levels have been validated using Genpathway’s Query assay, the differential binding observed between samples or between sites agreed very well with the ChIP Sequencing numbers, demonstrating good quantitation using this type of whole-genome assay.
Our IT department is overwhelmed with the prospect of the enormous amounts of data per run. Will handling of data on that scale be problematic?
Genpathway’s comprehensive analysis of ChIP Sequencing data condenses the results into manageable files that can be directly data-mined. While the raw data files are always sent to the customer as well, there is no need for the customer to deal with the large raw data files. In addition, Genpathway scientists are available for consultation regarding more complex analyses as desired.
What material should I send for the DNA methylation assay?
You can send purified DNA or frozen cells or tissues. If purified DNAs are sent, we ask that they be treated with RNase before the final concentrations are determined, and that 200-400 ng of each DNA be run on a 1% agarose gel to verify the DNA is of high molecular weight.
If cells or tissues are sent, they should not be fixed. Simply freeze the tissue or cell pellets and Genpathway will extract the DNA.
Do you have a preference between DNA or cells/tissue for the DNA methylation assay?
This is up to the customer, but if there is a choice, we prefer that cells or tissue are sent so Genpathway can extract the DNA. If DNA is sent, Genpathway still goes through a clean-up process with RNase and DNA purification followed by a final determination of DNA concentration. Results from this additional processing are also sent to the customer.
How do Genpathway’s ChIP/DNA IP protocols compare with commercially available ChIP kits?
Genpathway has optimized its protocols for better sensitivity and specificity (the latter referring to pull-down of significantly less background DNA). Recently, a direct comparison of Genpathway’s protocols with 5 ChIP kits showed that Genpathway’s protocols performed in a significantly superior manner. You may request more information about the comparison from Genpathway.
© 2010 Genpathway, Inc.
Chromatin and Methylated DNA Immunoprecipitation
"ChIP" Products & Services
"ChIP" Products & Services
