ISSN: 1511-3701 Pertanika J. Trop. Agric. Sci. 34 (2): 373 - 380 (2011) © Universiti Putra Malaysia Press Received: 2 September 2010 Accepted: 23 September 2010 * Corresponding Author INTRODUCTION Apoptosis or programmed cell death is defned as a mechanism of cellular suicide which occurs after sufficient cellular damage. Apoptosis differs from necrosis and it is involved in the development of many diseases, such as cancer (Arends & Wyllie, 1991; Nikitakis et al., 2004; Loro et al., 2003; 2005). Various imbalances in the apoptotic system, such as insufficient Detection of Bcl-2 Gene in Leukaemic Rats Using an EvaGreen Real-time RT-PCR Assay H. Nursyuhada 1 , H. Hazilawati 1* , A.H. Hutheyfa 1 , S.M. Rosly 2 , S. Shanmugavellu 2 , M.M. Noordin 1 and S. Jasni 1 1 Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia 2 Strategic Livestock Research Centre, Malaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor, Malaysia * E-mail: hazila@vet.upm.edu.my ABSTRACT Bcl-2 is an anti-apoptotic gene that is involved in the apoptosis process. Suppression of apoptosis by anti- apoptotic gene can contribute to the occurrence of diseases such as leukaemia. The objectives of this study were 2-folds: frst, to compare the sensitivity of an EvaGreen quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) with a conventional RT-PCR for the amplifcation of the Bcl-2 gene; second, to determine the expression of the Bcl-2 gene in N-methyl-N-nitrosourea (MNU)-induced leukaemia in rats using the EvaGreen qRT-PCR assay. A total of 32 male Sprague Dawley rats were assigned into two groups (n=16), namely, control and MNU groups. In particular, MNU was administered intraperitoneally (i.p) at a dose of 60 mg/kg body weight per injection at two times per week for 2 consecutive weeks. The rats were sacrifced after fve months and blood samples were collected for RNA extraction and haemogram. The RNAs were converted into cDNA and amplifed using both the EvaGreen qPCR and the conventional PCR assays. All the results were normalised with a housekeeper gene, i.e. glyceraldehyde 3-phosphate dehydrogenase (GADPH). The products of amplifcation were run on gel electrophoresis and all the results were then compared. Based on the relative intensity of the bands, the EvaGreen qRT-PCR assay was highly sensitive compared to the conventional RT-PCR assay as the Bcl-2 gene could not be amplifed using the conventional RT-PCR. Interestingly, the results in this study showed that the expression of Bcl-2 was higher in rats with marked lymphocytosis as compared to the leukaemic rats with normal to mildly increase in lymphocyte count. In conclusion, EvaGreen qRT-PCR assay is more sensitive compared to the conventional RT-PCR, and Bcl-2 gene is abundantly expressed in leukaemic rats with marked lymphocytosis compared to the leukaemic rats with normal to mildly increase in lymphocyte number. Keywords: Bcl-2, leukaemia, N-methyl-N-nitrosourea (MNU), EvaGreen qRT-PCR, conventional RT- PCR assays brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Universiti Putra Malaysia Institutional Repository