Differential Display RT-PCR And Suppression Subtractive Hybridization Identified Genes Differentially Expressed Between Untreated And Relapsed Acute Leukemia

Objective Differences in gene expression are likely to explain the phenotypic differences between untreated and relapsed acute leukemias (AL). To identify the genes differentially expressed between them and to isolate relapse-related genes of leukemia, differential display reverse transcription polymerase chain reaction (DDRT-PCR) and/or suppression subtractive hybridization (SSH) were used to isolate and identify the novel cDNA expression sequence tags (EST) from untreated and relapsed marrows of the same AL patient. Full cDNA sequences can be acquired from these novel ESTs by RACE, which will provide important information about relapse mechanism of leukemia.Methods From untreated and relapsed marrows of 3 AL patients, total RNA were isolated and purified. DDRT-PCR method with silver staining and SSH method were used to isolate differential ESTs from untreated and relapsed marrows of the same patient. The expression sequence tags were ligated to T-vector and transformed into E.coli competent cells. Theplasmid DNA were sequenced and compared with data in GenBank by BLASTN. According to the sequences of some novel ESTs, the primers were designed and synthesized. Differential expressions of these novel genes were analyzed by reverse transcription-PCR (RT-PCR). 110 AL patients were divided into two groups: untreated (newly diagnosed) and relapsed patients. The expression levels of these novel genes were detected by RT-PCR in peripheral blood or bone marrow samples from AL patients.Results 22 novel ESTs differentially expressed between untreated and relapsed AL samples were screened out. Among them, 8 ESTs were registered in GenBank dbEST database. The accession numbers in GenBank were CB331913, CB412263, CB412260, CB412262, CB412259, CB412265, CB412264 and CB412261 respectively. RT-PCR analyses demonstrated that expression level of a novel gene (DD-86) was much higher in the relapsed AL patients than in the untreated AL patients, but another novel gene (SSH-102) was expressed on much higher levels in the untreated patients than in the relapsed AL patients. They may be novelly relapse-related genes in AL.Conclusions Through the study mentioned above, we can draw following conclusion: 1. 22 novel ESTs differentially expressed between untreated and relapsed AL samples have been screened out by DDRT-PCR and SSH method. 2 ESTs have been confirmed to be related with the relapse of AL by RT-PCR. 2. Compared with DDRT-PCR method, SSH is better for rapid screening of the differentially expressed genes. It is more simple, sensitive , reproducible and predominantly lacks false positiveness. Italso has more information. Thereby it drastically reduces the workload on the experimenter. 3. In order to study the molecular mechanism for relapse of AL and to screen out specifically relapse-related genes, further studies about these novel genes will be carried out, including analyses of genes specificity and their function and acquisition of full cDNA.