Detection of BRAF V600E Mutation in Thyroid Tissue
Detection of BRAF V600E Mutation in Thyroid Tissue
One hundred patients diagnosed with PTC were enrolled in the study, and informed consent was obtained. The study was approved by the Gangnam Severance Hospital (Seoul, Korea) institutional review board. DNA was extracted from paraffin block using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Elution volume was 40 μL. To evaluate the limit of detecting mutants among wild-types, wild-type DNA (HeLa cell line) was mixed with mutant DNA (Colo205 cancer cell line). The relative concentrations (wild-type: mutant) of DNA were 0:100, 50:50, 80:20, 90:10, 95:5, 99:1, 99.5:0.05, and 100:0.
We used the PNAClamp BRAF mutation detection kit (Panagene, Daejeon, Korea) to detect the BRAF V600E mutation. All reactions totaled 20 μL in volume and contained template DNA, primers, a PNA probe, and SYBR green PCR master mix. Real-time PCR reaction of PNA-clamping PCR was performed using a CFX96 real-time PCR system (Bio-Rad, Pleasanton, CA). PCR cycling conditions included a 5-minute hold at 94°C followed by 40 cycles of 30 seconds at 94°C, 20 seconds at 70°C, 30 seconds at 63°C, and 30 seconds at 72°C.
In this assay, the PNA probe and primers were separate oligonucleotides, and the PNA probe was located between forward and reverse primers in the template. Positive signals were detected by intercalation of SYBR green fluorescent dye. The PNA probe sequences, which are complementary to wild-type (V600), enhance preferential amplification of mutant sequences by competitively inhibiting amplification of wild-type sequences (Figure 1). PCR efficiency was determined by measuring the cycle threshold (CT) value. CT values for the control and mutation assays were obtained by observing the SYBR green amplification plots. The delta CT (ΔCT) value was calculated by subtracting the CT value of a tested sample from the standard CT value of a clamping control sample ([Standard CT] – [Sample CT] = ΔCT). The cutoff for a positive sample was ΔCT = 2.
The BRAF V600E mutation was also detected using the Anyplex BRAF V600E real-time detection system (Seegene, Seoul, Korea). For 1 reaction, the mixture contained 2 μL of ×10 BRAF Oligo Mix, 3 μL of 8-Mop (8-methoxypsoralen) solution to prevent carryover contamination, and 10 μL of Anyplex PCR master mix containing DNA polymerase, buffer, and deoxynucleoside triphosphates. The reaction tube was either inverted 5 times or quickly vortexed to mix materials. Fifteen microliters of the reaction mixture was dispensed into 0.2-mL PCR tubes. Five microliters of each sample's nucleic acid was added to the reaction mixture tube to bring the total reaction volume to 20 μL. Real-time PCR was performed on a CFX96 real-time PCR system or StepOne real-time PCR systems (Applied Biosystems, Carlsbad, CA) under the following conditions: hold for 15 minutes at 95°C, followed by 45 cycles of 30 seconds at 95°C and 30 seconds at 60°C.
The CT in real-time PCR assay is defined as the number of cycles at which the fluorescent signal exceeds the threshold. A sample and internal control with a CT value below 40 were considered positive. Each run contained a positive control and negative control.
Quantitative estimation of BRAF V600E mutants using pyrosequencing was performed as described previously. Briefly, PCR amplification was performed with a forward primer (5'-GAAGACCTCACAGTAAAAATAG-3') and a reverse primer (5'-biotin-ATAGCCTCAATTCTTAC-CATCC-3') on a PTC-200 thermal cycler (MJ Research, Waltham, MA). The pyrosequencing reaction was done with a sequencing primer (5'-biotin-ATAGCCTCAATTCTTAC-CATCC-3') on a Pyromark Q24 instrument (Qiagen). Pyrogram outputs were analyzed with the PyroMark Q24 software (Qiagen) to determine the percentage of mutant vs wild-type alleles according to relative peak height.
The Student t test was used to analyze differences in mean age and mean tumor size between the BRAF V600E–positive and –negative groups. Differences in multifocality, extrathyroidal invasion, and lymph node metastasis were analyzed with a χ test. Differences in stage were analyzed with the Fisher exact test. Univariate and multivariate logistic regression analyses were performed to determine the association between BRAF V600E mutation and clinicopathologic features. SPSS version 12.0 (SPSS, Chicago, IL) was used for the statistical analysis.
Materials and Methods
One hundred patients diagnosed with PTC were enrolled in the study, and informed consent was obtained. The study was approved by the Gangnam Severance Hospital (Seoul, Korea) institutional review board. DNA was extracted from paraffin block using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Elution volume was 40 μL. To evaluate the limit of detecting mutants among wild-types, wild-type DNA (HeLa cell line) was mixed with mutant DNA (Colo205 cancer cell line). The relative concentrations (wild-type: mutant) of DNA were 0:100, 50:50, 80:20, 90:10, 95:5, 99:1, 99.5:0.05, and 100:0.
PNA-mediated Clamping PCR
We used the PNAClamp BRAF mutation detection kit (Panagene, Daejeon, Korea) to detect the BRAF V600E mutation. All reactions totaled 20 μL in volume and contained template DNA, primers, a PNA probe, and SYBR green PCR master mix. Real-time PCR reaction of PNA-clamping PCR was performed using a CFX96 real-time PCR system (Bio-Rad, Pleasanton, CA). PCR cycling conditions included a 5-minute hold at 94°C followed by 40 cycles of 30 seconds at 94°C, 20 seconds at 70°C, 30 seconds at 63°C, and 30 seconds at 72°C.
In this assay, the PNA probe and primers were separate oligonucleotides, and the PNA probe was located between forward and reverse primers in the template. Positive signals were detected by intercalation of SYBR green fluorescent dye. The PNA probe sequences, which are complementary to wild-type (V600), enhance preferential amplification of mutant sequences by competitively inhibiting amplification of wild-type sequences (Figure 1). PCR efficiency was determined by measuring the cycle threshold (CT) value. CT values for the control and mutation assays were obtained by observing the SYBR green amplification plots. The delta CT (ΔCT) value was calculated by subtracting the CT value of a tested sample from the standard CT value of a clamping control sample ([Standard CT] – [Sample CT] = ΔCT). The cutoff for a positive sample was ΔCT = 2.
Real-time PCR Using Anyplex BRAF V600E Real-time Detection System
The BRAF V600E mutation was also detected using the Anyplex BRAF V600E real-time detection system (Seegene, Seoul, Korea). For 1 reaction, the mixture contained 2 μL of ×10 BRAF Oligo Mix, 3 μL of 8-Mop (8-methoxypsoralen) solution to prevent carryover contamination, and 10 μL of Anyplex PCR master mix containing DNA polymerase, buffer, and deoxynucleoside triphosphates. The reaction tube was either inverted 5 times or quickly vortexed to mix materials. Fifteen microliters of the reaction mixture was dispensed into 0.2-mL PCR tubes. Five microliters of each sample's nucleic acid was added to the reaction mixture tube to bring the total reaction volume to 20 μL. Real-time PCR was performed on a CFX96 real-time PCR system or StepOne real-time PCR systems (Applied Biosystems, Carlsbad, CA) under the following conditions: hold for 15 minutes at 95°C, followed by 45 cycles of 30 seconds at 95°C and 30 seconds at 60°C.
The CT in real-time PCR assay is defined as the number of cycles at which the fluorescent signal exceeds the threshold. A sample and internal control with a CT value below 40 were considered positive. Each run contained a positive control and negative control.
Pyrosequencing
Quantitative estimation of BRAF V600E mutants using pyrosequencing was performed as described previously. Briefly, PCR amplification was performed with a forward primer (5'-GAAGACCTCACAGTAAAAATAG-3') and a reverse primer (5'-biotin-ATAGCCTCAATTCTTAC-CATCC-3') on a PTC-200 thermal cycler (MJ Research, Waltham, MA). The pyrosequencing reaction was done with a sequencing primer (5'-biotin-ATAGCCTCAATTCTTAC-CATCC-3') on a Pyromark Q24 instrument (Qiagen). Pyrogram outputs were analyzed with the PyroMark Q24 software (Qiagen) to determine the percentage of mutant vs wild-type alleles according to relative peak height.
Statistics
The Student t test was used to analyze differences in mean age and mean tumor size between the BRAF V600E–positive and –negative groups. Differences in multifocality, extrathyroidal invasion, and lymph node metastasis were analyzed with a χ test. Differences in stage were analyzed with the Fisher exact test. Univariate and multivariate logistic regression analyses were performed to determine the association between BRAF V600E mutation and clinicopathologic features. SPSS version 12.0 (SPSS, Chicago, IL) was used for the statistical analysis.
