It is a group for which not only the HLA phenotypeIt is a group for

It is a group for which not only the HLA phenotype
It is a group for which not only the HLA phenotype is available, but also the genotype, that is, the haplotypes were determined in each case, particularly in regard to class II genes.Typing of bcr-abl fusion transcripts by reversetranscriptase PCR Leukocytes prepared by erythrocyte lysis of bone marrow aspirate (1? ml) and peripheral blood (10 ml) samples collected in ethylene diamine tetraacetic acid were used for RNA isolation. Total RNA was isolated by using Trizol reagent (Invitrogen Lifetechnologies, USA) according to the manufacturer’s instruction. The integrity of RNA was determined by gel electrophoresis prior to reverse transcription (RT). Total RNA (1? ) from samples with intact 28 s and 18 s RNA was converted to cDNA by using random hexamers and Superscript II reverse transcriptase (Invitrogen Lifetechnologies) according to the recommendations of the manufacturer.Each sample was amplified in duplicate for bcr-abl in a multiplex GW9662 chemical information RT-PCR using an abl primer in combination with bcr b2- and e1-specific primers [17]. The t(9;22)-positive cell lines KBM7, K562, and B15, which carry b2a2, b3a2, and e1a2 fusion genes, respectively, served as positive controls. [18,19] The HL60 cell line was used as negative control.HLA typing HLA typing was performed by molecular methods. For class I, HLA typing was done at the intermediate-resolution level by using enzyme linked probe hybridizationPage 2 of(page number not for citation purposes)BMC Cancer 2004,http://www.biomedcentral.com/1471-2407/4/assay with sequence-specific oligonucleotide probe (ELPHA-SSOP) (Biotest, Germany). The sequence-specific oligonucleotide probes were used to identify polymorphic sequence motifs. The hybridization between probe and target DNA from the series of amplified PCR products was detected by a method adapted from the protein enzyme linked immunosorbent assay (ELISA) technique. For class II, HLA typing was done at the high-resolution level by using the sequence specific primers (SSP) (Genovision, USA or Pel-freez, USA). The technique uses a battery of known sequence specific primers to amplify specific alleles or group of alleles. This typing method is based on the fact that a completely matched primer will be used more efficiently in the PCR reaction than a partially mismatched primer. The electrophoresis bands generated were compared with the kit standards.Statistical analysis HLA gene frequencies (not phenotype frequency) were calculated in two different populations: (1) Healthy individuals typed as potential candidates for donating bone marrow; and (2) Patients diagnosed as having CML or ALL in whom bcr-abl transcripts were identified.Table 1:HEALTHY DONORS* HLA Class I: HLA Class II: 752CML + ALL* b2a2: b3a2: e1a2: 192 208*Numbers of HLA genes available in the different populations to calculate HLA allele frequencies (not the number of individuals in these populations taking into account that an individual carries two genes for each loci).DAEALQRPVAS [10,15], ATGFKQSSKALQRPVAS [23], and IPLTINKEEALQRPVAS [24].Results and discussionOur findings are summarized in Tables 2,3,4, which include only statistically significant observations with 2 > 3.84, corresponding to a significance level of p < 0.05. Relative risk > 1 indicates that a particular HLA allele is more frequent in a patient population with a particular transcript than within the corresponding healthy population. Relative risk PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26780312 < 1 indicates a negative association, with a gene frequency significantly.