Int. J. Mol. Sci. 2013, 14(2), 3456-3466; doi:10.3390/ijms14023456
Article

Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction

1 Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen 4032, Hungary 2 Thrombosis and Haemostasis Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen 4032, Hungary 3 Department of Internal Medicine, University of Debrecen, Medical and Health Science Center, Debrecen 4032, Hungary
* Author to whom correspondence should be addressed.
Received: 31 December 2012; in revised form: 21 January 2013 / Accepted: 30 January 2013 / Published: 6 February 2013
(This article belongs to the Section Molecular Diagnostics)
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Abstract: RT-qPCR is the standard method for studying changes in relative transcript level in different experimental and clinical conditions and in different tissues. No validated reference genes have been reported for the normalization of transcript level in platelets. The very low level of platelet RNA and the elimination of leukocyte contamination represented special methodological difficulties. Our aims were to apply a simple technique to separate platelets for transcript level studies, and select the most stable reference genes for platelets from healthy individuals and from patients with the history of myocardial infarction. We developed a simple, straightforward method of platelet separation for RNA isolation. Platelet activation was inhibited by using acid-citrate-dextrose for anticoagulation and by prostaglandin E1. Leukocyte contamination was eliminated by three consecutive centrifugations. Samples prepared by this method were free of leukocytes, showed no inhibition in PCR reaction and no RNA degradation. The assay demands low blood volume, which complies with the requirements of everyday laboratory routine. Seventeen potential reference genes were investigated, but eight of them were excluded during optimization. The stability of the remaining genes, EEF2, EAR, ACTB, GAPDH, ANAPC5, OAZ1, HDGF, GNAS, and CFL1, were determined by four different descriptive statistics. GAPDH, GNAS, and ACTB were shown to be the most stable genes in platelets of healthy individuals, while HDGF, GNAS, and ACTB were the most stable in platelets of patients with the history of myocardial infarction. The results confirm that data normalization needs assessment of appropriate reference genes for a particular sample set.
Keywords: transcript level; normalization; platelet; reference gene; RT-qPCR

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MDPI and ACS Style

Zsóri, K.S.; Muszbek, L.; Csiki, Z.; Shemirani, A.H. Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction. Int. J. Mol. Sci. 2013, 14, 3456-3466.

AMA Style

Zsóri KS, Muszbek L, Csiki Z, Shemirani AH. Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction. International Journal of Molecular Sciences. 2013; 14(2):3456-3466.

Chicago/Turabian Style

Zsóri, Katalin S.; Muszbek, László; Csiki, Zoltán; Shemirani, Amir H. 2013. "Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction." Int. J. Mol. Sci. 14, no. 2: 3456-3466.

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