Validation of reference genes for the determination of platelet transcript level in healthy individuals and in patients with the history of myocardial infarction

Katalin S. Zsóri, L. Muszbek, Zoltán Csiki, Amir H. Shemirani

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3456-3466
Number of pages11
JournalInternational Journal of Molecular Sciences
Volume14
Issue number2
DOIs
Publication statusPublished - Feb 2013

Fingerprint

myocardial infarction
Platelets
platelets
genes
Blood Platelets
Genes
History
Myocardial Infarction
histories
leukocytes
RNA
Leukocytes
Anaphase-Promoting Complex-Cyclosome Apc5 Subunit
contamination
Contamination
prostaglandins
Dextrose
Needs Assessment
blood volume
Alprostadil

Keywords

  • Normalization
  • Platelet
  • Reference gene
  • RT-qPCR
  • Transcript level

ASJC Scopus subject areas

  • Computer Science Applications
  • Molecular Biology
  • Catalysis
  • Inorganic Chemistry
  • Spectroscopy
  • Organic Chemistry
  • Physical and Theoretical Chemistry

Cite this

Validation of reference genes for the determination of platelet transcript level in healthy individuals and in patients with the history of myocardial infarction. / Zsóri, Katalin S.; Muszbek, L.; Csiki, Zoltán; Shemirani, Amir H.

In: International Journal of Molecular Sciences, Vol. 14, No. 2, 02.2013, p. 3456-3466.

Research output: Contribution to journalArticle

@article{a826a0b2ea1d45f7ac8f9d12e01680a1,
title = "Validation of reference genes for the determination of platelet transcript level in healthy individuals and in patients with the history of myocardial infarction",
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 = "Normalization, Platelet, Reference gene, RT-qPCR, Transcript level",
author = "Zs{\'o}ri, {Katalin S.} and L. Muszbek and Zolt{\'a}n Csiki and Shemirani, {Amir H.}",
year = "2013",
month = "2",
doi = "10.3390/ijms14023456",
language = "English",
volume = "14",
pages = "3456--3466",
journal = "International Journal of Molecular Sciences",
issn = "1661-6596",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "2",

}

TY - JOUR

T1 - Validation of reference genes for the determination of platelet transcript level in healthy individuals and in patients with the history of myocardial infarction

AU - Zsóri, Katalin S.

AU - Muszbek, L.

AU - Csiki, Zoltán

AU - Shemirani, Amir H.

PY - 2013/2

Y1 - 2013/2

N2 - 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.

AB - 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.

KW - Normalization

KW - Platelet

KW - Reference gene

KW - RT-qPCR

KW - Transcript level

UR - http://www.scopus.com/inward/record.url?scp=84875089327&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84875089327&partnerID=8YFLogxK

U2 - 10.3390/ijms14023456

DO - 10.3390/ijms14023456

M3 - Article

C2 - 23389042

AN - SCOPUS:84875089327

VL - 14

SP - 3456

EP - 3466

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 2

ER -