Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay

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: first, to compare the sensitivity of an EvaGreen quantitative real-t...

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Main Authors: Haron, Nursyuhada, Hamzah, Hazilawati, Al-Salih, Hutheyfa A. H., Shaari, Mohd Rosly, Sithambaram, Shanmugavelu, Mohamed Mustapha, Noordin, Sabri, Jasni
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Language:English
Published: Universiti Putra Malaysia Press 2011
Online Access:http://psasir.upm.edu.my/id/eprint/25343/1/24%20Pg%20373-380.pdf
http://psasir.upm.edu.my/id/eprint/25343/
http://www.pertanika.upm.edu.my/Pertanika%20PAPERS/JTAS%20Vol.%2034%20(2)%20Aug.%202011/24%20Pg%20373-380.pdf
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spelling my.upm.eprints.253432016-11-21T05:29:52Z http://psasir.upm.edu.my/id/eprint/25343/ Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay Haron, Nursyuhada Hamzah, Hazilawati Al-Salih, Hutheyfa A. H. Shaari, Mohd Rosly Sithambaram, Shanmugavelu Mohamed Mustapha, Noordin Sabri, Jasni 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: first, to compare the sensitivity of an EvaGreen quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) with a conventional RT-PCR for the amplification of the Bcl-2 gene; second, to determine the expression of the Bcl-2 gene in N-methyl-N-nitrosourea (MNU)-induced leukaemiain 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 sacrificed after five months and blood samples were collected for RNA extraction and haemogram. The RNAs were converted into cDNA and amplified 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 amplification 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 amplified 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. Universiti Putra Malaysia Press 2011 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/25343/1/24%20Pg%20373-380.pdf Haron, Nursyuhada and Hamzah, Hazilawati and Al-Salih, Hutheyfa A. H. and Shaari, Mohd Rosly and Sithambaram, Shanmugavelu and Mohamed Mustapha, Noordin and Sabri, Jasni (2011) Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay. Pertanika Journal of Tropical Agricultural Science, 34 (2). pp. 373-380. ISSN 1511-3701; ESSN: 2231-8542 http://www.pertanika.upm.edu.my/Pertanika%20PAPERS/JTAS%20Vol.%2034%20(2)%20Aug.%202011/24%20Pg%20373-380.pdf
institution Universiti Putra Malaysia
building UPM Library
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country Malaysia
content_provider Universiti Putra Malaysia
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language English
description 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: first, to compare the sensitivity of an EvaGreen quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) with a conventional RT-PCR for the amplification of the Bcl-2 gene; second, to determine the expression of the Bcl-2 gene in N-methyl-N-nitrosourea (MNU)-induced leukaemiain 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 sacrificed after five months and blood samples were collected for RNA extraction and haemogram. The RNAs were converted into cDNA and amplified 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 amplification 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 amplified 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.
format Article
author Haron, Nursyuhada
Hamzah, Hazilawati
Al-Salih, Hutheyfa A. H.
Shaari, Mohd Rosly
Sithambaram, Shanmugavelu
Mohamed Mustapha, Noordin
Sabri, Jasni
spellingShingle Haron, Nursyuhada
Hamzah, Hazilawati
Al-Salih, Hutheyfa A. H.
Shaari, Mohd Rosly
Sithambaram, Shanmugavelu
Mohamed Mustapha, Noordin
Sabri, Jasni
Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay
author_facet Haron, Nursyuhada
Hamzah, Hazilawati
Al-Salih, Hutheyfa A. H.
Shaari, Mohd Rosly
Sithambaram, Shanmugavelu
Mohamed Mustapha, Noordin
Sabri, Jasni
author_sort Haron, Nursyuhada
title Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay
title_short Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay
title_full Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay
title_fullStr Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay
title_full_unstemmed Detection of Bcl-2 gene in leukaemic rats using an EvaGreen real-time RT-PCT assay
title_sort detection of bcl-2 gene in leukaemic rats using an evagreen real-time rt-pct assay
publisher Universiti Putra Malaysia Press
publishDate 2011
url http://psasir.upm.edu.my/id/eprint/25343/1/24%20Pg%20373-380.pdf
http://psasir.upm.edu.my/id/eprint/25343/
http://www.pertanika.upm.edu.my/Pertanika%20PAPERS/JTAS%20Vol.%2034%20(2)%20Aug.%202011/24%20Pg%20373-380.pdf
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score 13.222552