Author/Editor		Pajič, Tadej; Poje, Mateja; Komel, Radovan; Černelč, Peter
Title		Ugotavljanje specifičnega cDNA-segmenta za BCR-ABL mRNA-prepis z načinom obratnega prepisovanja in verižne reakcije s polimerazo pri bolnikih s kronično mieloično levkemijo
Translated title		Detection of specific BCR-ABL mRNA transcript in chronic myeloid leukemia patients by the reverse trancriptase and polymerase chain reaction
Type		članek
Source		Med Razgl
Vol. and No.		Letnik 39, št. Suppl 7
Publication year		2000
Volume		str. 1-9
Language		slo
Abstract		Background. For the great majority of patients with chronic myeloid leukaemia (CML), the Philadelphia (Ph) chromosome is a specific marker of the malignant clone. Molecular analy- sis of the translocation t (9,22) and knowledge the structure of fusion BCR-ABL gene have enabled the assessment and follow-up of the disease by specific molecular methods such as Southern blotting, fluorescence in situ hybridization and reverse transcriptase - polymerase chain reaction (RT-PCR). RT-PCR is a rapid and powerful method for detection of specific sequences and is suitable to follow-up the disease in the complete cytogenetic remission. Methods. RT-PCR was used to amplify to BCR-ABL cDNA after reverse transcription of the mRNA. PCR for BCR-ABL in the CML was typically performed in two separate reactions: one for b2a2 and/or b3a2 mRNA transcript and a second for a control ABL gene in order to demonstrate adequate eDNA quality. The nested PCR strategy was used to increase the specificity of the method. PCR products were analysed by electrophoresis in agarose gels, stained with ethidium bromide and detected under UV light. Results. We analysed samples from 18 patients with chronic myeloid leukaemia by RT-PCR. Specific BCR-ABL mRNA transcript was determined in 15 out of 18 analysed samples. We assessed b2a2 transcript in six (40%) and b3a2 in eight (53%) out of 15 BCR-ABL positive patients with CML. Both transcripts were established in one sample (6%). Conclusion. We successfully applied the PCR method to blood and bone marrow samples from patients with CML. The method is suitable for routine work and represents an additional tool that can be used for diagnosis and improved estimate success in medical treatment of patients with CML.
Summary		Izhodišča. Za veliko večino bolnikov s kronično mieloično levkemijo je Philadelphia kromosom specifični označevalec neoplastičnega klona. Molekularna določitev translokacije t (9,22) in poznavanje zgradbe hibridnih genov BCR-ABL sta omogočili ugotavljanje in spremljanje bolezni z molekularno genetičnimi načini, kot je hibridizacija po Southernu, fluorescenčna in situ hibridizacija (FISH) ter obratno prepisovanje in verižna reakcija s polimerazo (RT PCR). RT-PCR je hiter in občutljiv način za ugotavljanje specifičnih zaporedij in je primeren za spremljanje bolnikov v popolni citogenetični remisiji. Metode. Z načinom obratnega prepisovanja in verižne reakcije s polimerazo smo ugotavljali značilen cDNA-segment za BCR-ABL mRNA-prepis. Reakcijo smo izvedli ločeno: eno za prepis b3a2 in/ali b2a2 in drugo zaradi prikaza ustreznosti cDNA-molekule, za kontrolno zaporedje mRNA, ki nastane iz gena ABL. Za povečanje specifičnosti načina smo del pridelka prvega pomnoževanja BCR-ABL cDNA-segmenta ponovno pomnožili s pomočjo oligonukleotidov, ki so se prilegali znotraj prvega pridelka. Velikost pridellcov reakcij pomnoževanj smo ocenjevali z elektroforezo v agaroznem gelu, obarvanem z etidijevim bromidom pod UV-svetlobo. Rezultati. Z RT-PCR-načinom smo ocenili 18 vzorcev bolnikov s kronično mieloično levke- mijo. Značilen BCR-ABL mRNA-prepis smo določili v 15 izmed 18 preiskovanih vzorcev. Prisotnost prepisa b2a2 smo ugotovili pri šestih (40 %) in b3a2 pri osmih (53 %) od petnajstih BCR-ABL mRNA-pozitivnih bolnikov. Pri enem bolniku smo zasledili oba prepisa (6 %). Zaključek. Uspešno smo vpeljali RT-PCR-način na vzorcih periferne krvi in kostnega mozga pri bolnikih s KML in ugotovili, da je primeren za redno delo. Predstavlja dopolnilno prei- skavo pri ugotavljanju bolezni in pri oceni uspeha zdravljenja pri bolnikih s KML.
Descriptors		LEUKEMIA, MYELOID, CHRONIC PHILADELPHIA CHROMOSOME TRANSLOCATION (GENETICS) POLYMERASE CHAIN REACTION DNA, COMPLEMENTARY GENES, ABL RNA, MESSENGER