Provided by SNPs [20]. This is illustrated by the identification of genes associated with rheumatoid arthritis [21], narcolepsy [22] and Behcet’s disease [23] using genome-wide association studies based on microsatellites. Hence, a genome-wide association study was performed to identify candidate genes that are strongly associated with radiosensitivity in humans. The screen analyzed data from 23,244 microsatellites in 360 cancer patients who had undergone radiotherapy and been graded for normal tissue adverse reactions. Forty-seven markers were identified as being of interest, with a role for the involvement of SEMA3A in radiosensitivity suggested.to the method of Collins et al [24]. Concentrations of individual gDNA Peretinoin manufacturer samples were adjusted to 8 ng/L. An equal volume of each of 90 gDNA samples from the HGG was combined to generate the first set of pooled gDNA and termed HGG-1. Similarly, 90 gDNA samples from the LGG were pooled and termed LGG-1. A second set of pooled gDNA samples was also Chloroquine (diphosphate)MedChemExpress Chloroquine (diphosphate) prepared from 90 samples of the HGG and 90 samples of the LGG, and these were termed HGG-2 and LGG-2, respectively.Analysis of microsatellite markersMethodsGrading of patients with low and high grade radiosensitivitySince 2001, the RadGenomics project has enrolled more than 3000 patients who have undergone radiotherapy. All patients provided written informed consent to participate in this study, which was approved by the Institutional Review Board at the National Institute of Radiological Sciences and by each collaborating institution. The acute adverse reactions of individual patients up to three months after completion of radiotherapy were graded according to the National Cancer Institute’s Common Toxicity Criteria (NCICTC) version 2. We retrospectively selected 180 patients, who presented with a severe acute reaction of equal to or greater than grade 3, as a high-grade group (HGG). We also retrospectively selected 180 patients with less than or equal to grade 1 acute reaction on any end point, as a low-grade group (LGG). The assignment of patients to the LGG and HGG took into consideration their cancer type, age, gender, treatment type, and radiation dose (table 1).Preparation of pooled DNA samplesAll microsatellite markers and the methods for microsatellite analysis used in this study are described in Tamiya et al [21]. The genomic location of the microsatellite markers was investigated using the UCSC Genome Browser http://genome.ucsc.edu/cgi-bin/ hgGateway. PCR primers to amplify microsatellites were designed to anneal at 57 , with forward primers having a 5′ fluorescent label (6-FAM or HEX). PCR was performed using the GeneAmp PCR system 9700 (GE Healthcare, Amersham Place, UK) in 20 L containing 48 ng pooled DNA, 0.5 U AmpliTaq DNA polymerase, reaction buffer containing 1.5 mM MgCl2 (GE Healthcare, Amersham Place, UK), 5 M of each primer, and 0.25 mM of each dNTP in 96- or 384-well plates. PCR profile was as follows; 96 for 5 min, 57 for 1 min, 72 for 1 min; 40 cycles of 96 for 45 s, 57 for 45 s, 72 for 1 min. For microsatellite typing of individual samples, PCR was performed in 12 L containing 2 ng DNA, 0.25 U AmpliTaq Gold DNA polymerase (GE Healthcare, Amersham Place, UK), reaction buffer containing 1.5 mM MgCl2, 5 M of each primer, and 0.2 mM of each dNTP in 96- or 384-well plates and amplified as above. PCR products were denatured in Hi-Di formamide (GE Healthcare, Amersham Place, UK) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27864321 at 95 for 3 min and separated by capillary electr.Provided by SNPs [20]. This is illustrated by the identification of genes associated with rheumatoid arthritis [21], narcolepsy [22] and Behcet’s disease [23] using genome-wide association studies based on microsatellites. Hence, a genome-wide association study was performed to identify candidate genes that are strongly associated with radiosensitivity in humans. The screen analyzed data from 23,244 microsatellites in 360 cancer patients who had undergone radiotherapy and been graded for normal tissue adverse reactions. Forty-seven markers were identified as being of interest, with a role for the involvement of SEMA3A in radiosensitivity suggested.to the method of Collins et al [24]. Concentrations of individual gDNA samples were adjusted to 8 ng/L. An equal volume of each of 90 gDNA samples from the HGG was combined to generate the first set of pooled gDNA and termed HGG-1. Similarly, 90 gDNA samples from the LGG were pooled and termed LGG-1. A second set of pooled gDNA samples was also prepared from 90 samples of the HGG and 90 samples of the LGG, and these were termed HGG-2 and LGG-2, respectively.Analysis of microsatellite markersMethodsGrading of patients with low and high grade radiosensitivitySince 2001, the RadGenomics project has enrolled more than 3000 patients who have undergone radiotherapy. All patients provided written informed consent to participate in this study, which was approved by the Institutional Review Board at the National Institute of Radiological Sciences and by each collaborating institution. The acute adverse reactions of individual patients up to three months after completion of radiotherapy were graded according to the National Cancer Institute’s Common Toxicity Criteria (NCICTC) version 2. We retrospectively selected 180 patients, who presented with a severe acute reaction of equal to or greater than grade 3, as a high-grade group (HGG). We also retrospectively selected 180 patients with less than or equal to grade 1 acute reaction on any end point, as a low-grade group (LGG). The assignment of patients to the LGG and HGG took into consideration their cancer type, age, gender, treatment type, and radiation dose (table 1).Preparation of pooled DNA samplesAll microsatellite markers and the methods for microsatellite analysis used in this study are described in Tamiya et al [21]. The genomic location of the microsatellite markers was investigated using the UCSC Genome Browser http://genome.ucsc.edu/cgi-bin/ hgGateway. PCR primers to amplify microsatellites were designed to anneal at 57 , with forward primers having a 5′ fluorescent label (6-FAM or HEX). PCR was performed using the GeneAmp PCR system 9700 (GE Healthcare, Amersham Place, UK) in 20 L containing 48 ng pooled DNA, 0.5 U AmpliTaq DNA polymerase, reaction buffer containing 1.5 mM MgCl2 (GE Healthcare, Amersham Place, UK), 5 M of each primer, and 0.25 mM of each dNTP in 96- or 384-well plates. PCR profile was as follows; 96 for 5 min, 57 for 1 min, 72 for 1 min; 40 cycles of 96 for 45 s, 57 for 45 s, 72 for 1 min. For microsatellite typing of individual samples, PCR was performed in 12 L containing 2 ng DNA, 0.25 U AmpliTaq Gold DNA polymerase (GE Healthcare, Amersham Place, UK), reaction buffer containing 1.5 mM MgCl2, 5 M of each primer, and 0.2 mM of each dNTP in 96- or 384-well plates and amplified as above. PCR products were denatured in Hi-Di formamide (GE Healthcare, Amersham Place, UK) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27864321 at 95 for 3 min and separated by capillary electr.