Optimization of Multiplex RT-PCR for Leukemia Typing and Subtyping

Abstract

Molecular typization and subtypization of leukemia by RT-PCR, is important due to monitoring of minimal residual disease (MRD), during and after treatement of patients with acute leukemia In our experiments of PCR optimization, we used BIO-RAD procedure for multiplex RT-PCR screening and split-out PCR for leukemia subtypization. This method is adapted or optimized for Perkin Elmer Gene Amp 9600 thermal cycler and Qiagen HotStar Taq DNA polymera.se as well. According to manufacturer instructions, using of some other PCR engines require optimization of corresponding PCR parameters. We used Perkin Elmer 2400 thermal cycler and Hot Star Taq DNA polymera.se. Total RNA was extracted from whole blood specimens, obtained from Clinics of haematology-KCU Sarajevo, taken from patients with acute leukemia, transferred to cDNA, and amplificated in PE 2400. Detection of PCR products is performed by 1,5% agarose gel electrophoresis. Obtained optimal PCR parameters were: annealing temperature -65°C, number of cycles for both, first round PCR amplification and nested PCR amplification was 30, final concertation of MgCl in reaction mixture was 3,28 mM. Instead recommended amount of cDNA (5 µl) and Hot Star Taq DNA polymera.se (0,4 µl) we used amounts of 10 µl and 1 µl By using of this optimized PCR protocol we detected genetic aberration inv(16)(p 13; q22) and by using of split out PCR, subtype G(192 bp electrophoretic band)(CBFB / M YH11 fusion genes).