This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
Determination of the CpG Island Methylator Phenotype (CIMP) in colorectal cancer using MethyLight
Daniel J. Weisenberger
commented on 17 September, 2006
PROTOCOL CORRECTIONS: In the original text of the protocol, we incorrectly described: 1) the evaluation of bisulfite-converted DNA and 2) the preparation of the Oligo Mix used to simplify the preparation of MethyLight reactions. Revisions of these sections are described below. Bisulfite Conversion & DNA Recovery: The bisulfite conversion and recovery of bisulfite-converted DNA steps are performed using the Zymo EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer’s instructions. M.SssI-modified DNA is also treated with bisulfite for use as a methylated reference in MethyLight assays. 1. We typically perform a preliminary MethyLight control PCR reaction with a small amount bisulfite-converted sample using the ALU-C4 (Alu repeats) bisulfite control reaction. The bisulfite-converted DNA recovered using the Zymo protocol is in a final volume of ~10 µl. The 10 µl sample is then diluted to a 100 µl volume. [However, this dilution is not absolute and can be changed depending on the amount of starting material.] We typically use 2 µl of the 100 µl sample and evaluate the amount of bisulfite-DNA using the ALU-C4 bisulfite control reaction (specific for Alu repeats). The C(t) value generated from this 1:5 dilution (2 µl bisulfite-DNA sample + 8 µl water) will give an indication of the approximate bisulfite-converted DNA sample amounts, and the degree to which the bisulfite-DNA sample can be further diluted when performing MethyLight assays. Since the ALU-C4 reaction is highly sensitive and will generate low C(t) values, a sample that gives a C(t) = 22 is considered maximally diluted. However, it should be noted that lower C(t) values are always preferred to achieve the best possible data. An ALU-C4 C(t) value of less than 20 is usually desirable. For the CIMP analysis, we have selected five MethyLight reactions. If the C(t) values of the 1:5 test dilution are appropriate (although more input DNA amounts will give improved C(t) values), a final bisulfite-DNA volume of ≥ 300 µl after dilution will allow all five CIMP and control reactions to be analyzed in duplicate and allows for pipetting or other errors. 2. The bisulfite-converted M.SssI-DNA sample, also recovered in a 10 µl volume after recovery, should also be diluted to 100 µl and then diluted another 1:10 (to a 1 ml final volume). From this 1 ml sample, we typically remove 10 µl for use in each MethyLight reaction. MethyLight Primer/Probe Preparation: 1. The MethyLight primers and probes, since they are lyophilized after synthesis, need to be dissolved in sterile water. The forward and reverse primers are prepared at a concentration of 300 µM and the probe at a concentration of 100 µM. Small aliquots of the primers at these concentrations should be made to prevent repeated freeze/thaw events. 2. Dilute the primers/probe to a working stock of 6µM (primers) and 2µM (probe). Then use 1.5 µl of each primer and the probe in a 30 µl MethyLight reaction. This results in a final concentration of 0.3 µM primers and 0.1 µM probe in each 30 µl MethyLight reaction. 3. Alternatively, one can also prepare an Oligo Mix containing both primers and the probe. This is achieved by combining the forward primer, reverse primer and probe in one tube as an Oligo Mix: (4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer, and 4 µl of the 100 µM probe in a 600 µl total volume). The concentrations of the forward and reverse primers in the Oligo Mix are 2 µM and the probe concentration is 0.67 µM. The use of 4.5 µl of the Oligo Mix per 30 µL MethyLight reaction represents the combined volumes from each of the two individual primers and the probe. Oligo Mix Preparation: • Prepare the forward and reverse primers at a concentration of 300 µM and the probe at a concentration of 100 µM. • The forward primer, reverse primer and probe can be combined in one tube to prepare the Oligo Mix. This consists of 4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer and 4 µl of the 100 µM probe in a 600 µl total volume: Oligo Mix: Forward primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Reverse primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Probe: 100 µM stock; use 4µl; 0.67 µM conc. in Oligo Mix Water: add 588 µl TOTAL VOLUME: 600 µl •Use 4.5 µl of this Oligo Mix per 30 µl MethyLight reaction. After addition to the PCR reaction mixture, the forward/reverse primers are at a concentration of 0.3µM and the probe at 0.1 µM. PCR Reaction Set-Up: PCR Component (Final Concentration) 4.2 µl 25mM MgCl2 (3.5 mM) 3.0 µl 10X Buffer (1X) 3.0 µl 10X stabilizer (1X) 0.6 µl 10mM dNTPs (200 µM) 4.6 µl water 1.5 µl 6µM forward primer (0.3 µM)* 1.5 µl 6µM reverse primer (0.3 µM)* 1.5 µl 2µM probe (0.1 µM)* 0.1 µl Taq Gold Polymerase 10.0 µl DNA sample *Alternatively, an Oligo Mix can be prepared. The Oligo Mix contains the forward and reverse primers (both 2 µM) and the probe (0.67 µM). After using 4.5 µl Oligo Mix in a 30 µl MethyLight reaction, the final forward and reverse primer concentrations are 0.3 µM and the final probe concentration is 0.1 µM.
Daniel J. Weisenberger
commented on 17 September, 2006
PROTOCOL CORRECTIONS: In the original text of the protocol, we incorrectly described: 1) the evaluation of bisulfite-converted DNA and 2) the preparation of the Oligo Mix used to simplify the preparation of MethyLight reactions. Revisions of these sections are described below. Bisulfite Conversion & DNA Recovery: The bisulfite conversion and recovery of bisulfite-converted DNA steps are performed using the Zymo EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer’s instructions. M.SssI-modified DNA is also treated with bisulfite for use as a methylated reference in MethyLight assays. 1. We typically perform a preliminary MethyLight control PCR reaction with a small amount bisulfite-converted sample using the ALU-C4 (Alu repeats) bisulfite control reaction. The bisulfite-converted DNA recovered using the Zymo protocol is in a final volume of ~10 µl. The 10 µl sample is then diluted to a 100 µl volume. [However, this dilution is not absolute and can be changed depending on the amount of starting material.] We typically use 2 µl of the 100 µl sample and evaluate the amount of bisulfite-DNA using the ALU-C4 bisulfite control reaction (specific for Alu repeats). The C(t) value generated from this 1:5 dilution (2 µl bisulfite-DNA sample + 8 µl water) will give an indication of the approximate bisulfite-converted DNA sample amounts, and the degree to which the bisulfite-DNA sample can be further diluted when performing MethyLight assays. Since the ALU-C4 reaction is highly sensitive and will generate low C(t) values, a sample that gives a C(t) = 22 is considered maximally diluted. However, it should be noted that lower C(t) values are always preferred to achieve the best possible data. An ALU-C4 C(t) value of less than 20 is usually desirable. For the CIMP analysis, we have selected five MethyLight reactions. If the C(t) values of the 1:5 test dilution are appropriate (although more input DNA amounts will give improved C(t) values), a final bisulfite-DNA volume of ≥ 300 µl after dilution will allow all five CIMP and control reactions to be analyzed in duplicate and allows for pipetting or other errors. 2. The bisulfite-converted M.SssI-DNA sample, also recovered in a 10 µl volume after recovery, should also be diluted to 100 µl and then diluted another 1:10 (to a 1 ml final volume). From this 1 ml sample, we typically remove 10 µl for use in each MethyLight reaction. MethyLight Primer/Probe Preparation: 1. The MethyLight primers and probes, since they are lyophilized after synthesis, need to be dissolved in sterile water. The forward and reverse primers are prepared at a concentration of 300 µM and the probe at a concentration of 100 µM. Small aliquots of the primers at these concentrations should be made to prevent repeated freeze/thaw events. 2. Dilute the primers/probe to a working stock of 6µM (primers) and 2µM (probe). Then use 1.5 µl of each primer and the probe in a 30 µl MethyLight reaction. This results in a final concentration of 0.3 µM primers and 0.1 µM probe in each 30 µl MethyLight reaction. 3. Alternatively, one can also prepare an Oligo Mix containing both primers and the probe. This is achieved by combining the forward primer, reverse primer and probe in one tube as an Oligo Mix: (4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer, and 4 µl of the 100 µM probe in a 600 µl total volume). The concentrations of the forward and reverse primers in the Oligo Mix are 2 µM and the probe concentration is 0.67 µM. The use of 4.5 µl of the Oligo Mix per 30 µL MethyLight reaction represents the combined volumes from each of the two individual primers and the probe. Oligo Mix Preparation: • Prepare the forward and reverse primers at a concentration of 300 µM and the probe at a concentration of 100 µM. • The forward primer, reverse primer and probe can be combined in one tube to prepare the Oligo Mix. This consists of 4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer and 4 µl of the 100 µM probe in a 600 µl total volume: Oligo Mix: Forward primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Reverse primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Probe: 100 µM stock; use 4µl; 0.67 µM conc. in Oligo Mix Water: add 588 µl TOTAL VOLUME: 600 µl •Use 4.5 µl of this Oligo Mix per 30 µl MethyLight reaction. After addition to the PCR reaction mixture, the forward/reverse primers are at a concentration of 0.3µM and the probe at 0.1 µM. PCR Reaction Set-Up: PCR Component (Final Concentration) 4.2 µl 25mM MgCl2 (3.5 mM) 3.0 µl 10X Buffer (1X) 3.0 µl 10X stabilizer (1X) 0.6 µl 10mM dNTPs (200 µM) 4.6 µl water 1.5 µl 6µM forward primer (0.3 µM)* 1.5 µl 6µM reverse primer (0.3 µM)* 1.5 µl 2µM probe (0.1 µM)* 0.1 µl Taq Gold Polymerase 10.0 µl DNA sample *Alternatively, an Oligo Mix can be prepared. The Oligo Mix contains the forward and reverse primers (both 2 µM) and the probe (0.67 µM). After using 4.5 µl Oligo Mix in a 30 µl MethyLight reaction, the final forward and reverse primer concentrations are 0.3 µM and the final probe concentration is 0.1 µM.
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Jun, 2006
Community comments: 2
Metrics
Comments: 2
HTML Views: ...
Subject Areas
Genetics
Associated Publications
CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer
by Weisenberger, D.J. et al.
Nature Genetics (06 June, 2006)
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
Determination of the CpG Island Methylator Phenotype (CIMP) in colorectal cancer using MethyLight
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Jun, 2006
Community comments: 2
Metrics
Comments: 2
HTML Views: ...
Subject Areas
Genetics
Daniel J. Weisenberger
commented on 17 September, 2006
PROTOCOL CORRECTIONS: In the original text of the protocol, we incorrectly described: 1) the evaluation of bisulfite-converted DNA and 2) the preparation of the Oligo Mix used to simplify the preparation of MethyLight reactions. Revisions of these sections are described below. Bisulfite Conversion & DNA Recovery: The bisulfite conversion and recovery of bisulfite-converted DNA steps are performed using the Zymo EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer’s instructions. M.SssI-modified DNA is also treated with bisulfite for use as a methylated reference in MethyLight assays. 1. We typically perform a preliminary MethyLight control PCR reaction with a small amount bisulfite-converted sample using the ALU-C4 (Alu repeats) bisulfite control reaction. The bisulfite-converted DNA recovered using the Zymo protocol is in a final volume of ~10 µl. The 10 µl sample is then diluted to a 100 µl volume. [However, this dilution is not absolute and can be changed depending on the amount of starting material.] We typically use 2 µl of the 100 µl sample and evaluate the amount of bisulfite-DNA using the ALU-C4 bisulfite control reaction (specific for Alu repeats). The C(t) value generated from this 1:5 dilution (2 µl bisulfite-DNA sample + 8 µl water) will give an indication of the approximate bisulfite-converted DNA sample amounts, and the degree to which the bisulfite-DNA sample can be further diluted when performing MethyLight assays. Since the ALU-C4 reaction is highly sensitive and will generate low C(t) values, a sample that gives a C(t) = 22 is considered maximally diluted. However, it should be noted that lower C(t) values are always preferred to achieve the best possible data. An ALU-C4 C(t) value of less than 20 is usually desirable. For the CIMP analysis, we have selected five MethyLight reactions. If the C(t) values of the 1:5 test dilution are appropriate (although more input DNA amounts will give improved C(t) values), a final bisulfite-DNA volume of ≥ 300 µl after dilution will allow all five CIMP and control reactions to be analyzed in duplicate and allows for pipetting or other errors. 2. The bisulfite-converted M.SssI-DNA sample, also recovered in a 10 µl volume after recovery, should also be diluted to 100 µl and then diluted another 1:10 (to a 1 ml final volume). From this 1 ml sample, we typically remove 10 µl for use in each MethyLight reaction. MethyLight Primer/Probe Preparation: 1. The MethyLight primers and probes, since they are lyophilized after synthesis, need to be dissolved in sterile water. The forward and reverse primers are prepared at a concentration of 300 µM and the probe at a concentration of 100 µM. Small aliquots of the primers at these concentrations should be made to prevent repeated freeze/thaw events. 2. Dilute the primers/probe to a working stock of 6µM (primers) and 2µM (probe). Then use 1.5 µl of each primer and the probe in a 30 µl MethyLight reaction. This results in a final concentration of 0.3 µM primers and 0.1 µM probe in each 30 µl MethyLight reaction. 3. Alternatively, one can also prepare an Oligo Mix containing both primers and the probe. This is achieved by combining the forward primer, reverse primer and probe in one tube as an Oligo Mix: (4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer, and 4 µl of the 100 µM probe in a 600 µl total volume). The concentrations of the forward and reverse primers in the Oligo Mix are 2 µM and the probe concentration is 0.67 µM. The use of 4.5 µl of the Oligo Mix per 30 µL MethyLight reaction represents the combined volumes from each of the two individual primers and the probe. Oligo Mix Preparation: • Prepare the forward and reverse primers at a concentration of 300 µM and the probe at a concentration of 100 µM. • The forward primer, reverse primer and probe can be combined in one tube to prepare the Oligo Mix. This consists of 4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer and 4 µl of the 100 µM probe in a 600 µl total volume: Oligo Mix: Forward primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Reverse primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Probe: 100 µM stock; use 4µl; 0.67 µM conc. in Oligo Mix Water: add 588 µl TOTAL VOLUME: 600 µl •Use 4.5 µl of this Oligo Mix per 30 µl MethyLight reaction. After addition to the PCR reaction mixture, the forward/reverse primers are at a concentration of 0.3µM and the probe at 0.1 µM. PCR Reaction Set-Up: PCR Component (Final Concentration) 4.2 µl 25mM MgCl2 (3.5 mM) 3.0 µl 10X Buffer (1X) 3.0 µl 10X stabilizer (1X) 0.6 µl 10mM dNTPs (200 µM) 4.6 µl water 1.5 µl 6µM forward primer (0.3 µM)* 1.5 µl 6µM reverse primer (0.3 µM)* 1.5 µl 2µM probe (0.1 µM)* 0.1 µl Taq Gold Polymerase 10.0 µl DNA sample *Alternatively, an Oligo Mix can be prepared. The Oligo Mix contains the forward and reverse primers (both 2 µM) and the probe (0.67 µM). After using 4.5 µl Oligo Mix in a 30 µl MethyLight reaction, the final forward and reverse primer concentrations are 0.3 µM and the final probe concentration is 0.1 µM.
Daniel J. Weisenberger
commented on 17 September, 2006
PROTOCOL CORRECTIONS: In the original text of the protocol, we incorrectly described: 1) the evaluation of bisulfite-converted DNA and 2) the preparation of the Oligo Mix used to simplify the preparation of MethyLight reactions. Revisions of these sections are described below. Bisulfite Conversion & DNA Recovery: The bisulfite conversion and recovery of bisulfite-converted DNA steps are performed using the Zymo EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer’s instructions. M.SssI-modified DNA is also treated with bisulfite for use as a methylated reference in MethyLight assays. 1. We typically perform a preliminary MethyLight control PCR reaction with a small amount bisulfite-converted sample using the ALU-C4 (Alu repeats) bisulfite control reaction. The bisulfite-converted DNA recovered using the Zymo protocol is in a final volume of ~10 µl. The 10 µl sample is then diluted to a 100 µl volume. [However, this dilution is not absolute and can be changed depending on the amount of starting material.] We typically use 2 µl of the 100 µl sample and evaluate the amount of bisulfite-DNA using the ALU-C4 bisulfite control reaction (specific for Alu repeats). The C(t) value generated from this 1:5 dilution (2 µl bisulfite-DNA sample + 8 µl water) will give an indication of the approximate bisulfite-converted DNA sample amounts, and the degree to which the bisulfite-DNA sample can be further diluted when performing MethyLight assays. Since the ALU-C4 reaction is highly sensitive and will generate low C(t) values, a sample that gives a C(t) = 22 is considered maximally diluted. However, it should be noted that lower C(t) values are always preferred to achieve the best possible data. An ALU-C4 C(t) value of less than 20 is usually desirable. For the CIMP analysis, we have selected five MethyLight reactions. If the C(t) values of the 1:5 test dilution are appropriate (although more input DNA amounts will give improved C(t) values), a final bisulfite-DNA volume of ≥ 300 µl after dilution will allow all five CIMP and control reactions to be analyzed in duplicate and allows for pipetting or other errors. 2. The bisulfite-converted M.SssI-DNA sample, also recovered in a 10 µl volume after recovery, should also be diluted to 100 µl and then diluted another 1:10 (to a 1 ml final volume). From this 1 ml sample, we typically remove 10 µl for use in each MethyLight reaction. MethyLight Primer/Probe Preparation: 1. The MethyLight primers and probes, since they are lyophilized after synthesis, need to be dissolved in sterile water. The forward and reverse primers are prepared at a concentration of 300 µM and the probe at a concentration of 100 µM. Small aliquots of the primers at these concentrations should be made to prevent repeated freeze/thaw events. 2. Dilute the primers/probe to a working stock of 6µM (primers) and 2µM (probe). Then use 1.5 µl of each primer and the probe in a 30 µl MethyLight reaction. This results in a final concentration of 0.3 µM primers and 0.1 µM probe in each 30 µl MethyLight reaction. 3. Alternatively, one can also prepare an Oligo Mix containing both primers and the probe. This is achieved by combining the forward primer, reverse primer and probe in one tube as an Oligo Mix: (4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer, and 4 µl of the 100 µM probe in a 600 µl total volume). The concentrations of the forward and reverse primers in the Oligo Mix are 2 µM and the probe concentration is 0.67 µM. The use of 4.5 µl of the Oligo Mix per 30 µL MethyLight reaction represents the combined volumes from each of the two individual primers and the probe. Oligo Mix Preparation: • Prepare the forward and reverse primers at a concentration of 300 µM and the probe at a concentration of 100 µM. • The forward primer, reverse primer and probe can be combined in one tube to prepare the Oligo Mix. This consists of 4 µl of the 300 µM forward primer, 4 µl of the 300 µM reverse primer and 4 µl of the 100 µM probe in a 600 µl total volume: Oligo Mix: Forward primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Reverse primer: 300 µM stock; use 4µl; 2 µM conc. in Oligo Mix. Probe: 100 µM stock; use 4µl; 0.67 µM conc. in Oligo Mix Water: add 588 µl TOTAL VOLUME: 600 µl •Use 4.5 µl of this Oligo Mix per 30 µl MethyLight reaction. After addition to the PCR reaction mixture, the forward/reverse primers are at a concentration of 0.3µM and the probe at 0.1 µM. PCR Reaction Set-Up: PCR Component (Final Concentration) 4.2 µl 25mM MgCl2 (3.5 mM) 3.0 µl 10X Buffer (1X) 3.0 µl 10X stabilizer (1X) 0.6 µl 10mM dNTPs (200 µM) 4.6 µl water 1.5 µl 6µM forward primer (0.3 µM)* 1.5 µl 6µM reverse primer (0.3 µM)* 1.5 µl 2µM probe (0.1 µM)* 0.1 µl Taq Gold Polymerase 10.0 µl DNA sample *Alternatively, an Oligo Mix can be prepared. The Oligo Mix contains the forward and reverse primers (both 2 µM) and the probe (0.67 µM). After using 4.5 µl Oligo Mix in a 30 µl MethyLight reaction, the final forward and reverse primer concentrations are 0.3 µM and the final probe concentration is 0.1 µM.
Associated Publications
CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer
by Weisenberger, D.J. et al.
Nature Genetics (06 June, 2006)