Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 50: 2271-2278, 2004. First published October 7, 2004; 10.1373/clinchem.2004.035386
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
clinchem.2004.035386v1
50/12/2271    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (16)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rox, J. M.
Right arrow Articles by Pötzsch, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rox, J. M.
Right arrow Articles by Pötzsch, B.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Hemostasis and Thrombosis
(Clinical Chemistry. 2004;50:2271-2278.)
© 2004 American Association for Clinical Chemistry, Inc.

Hemostasis and Thrombosis

Gene Expression Analysis in Platelets from a Single Donor: Evaluation of a PCR-Based Amplification Technique

Jutta Maria Rox1,1, Peter Bugert2,1, Jens Müller1, Alexander Schorr1, Peter Hanfland1, Katharina Madlener3, Harald Klüter2 and Bernd Pötzsch1,a

1 Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany.
2 Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, University of Heidelberg, Faculty of Clinical Medicine, Mannheim, Germany.
3 Department of Haemostaseology, Clinical Immunology and Transfusion Medicine, Kerckhoff-Klinik, Bad Nauheim, Germany.

aAddress correspondence to this author at: Institute of Experimental Haematology and Transfusion Medicine, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany. Fax 49-228-287-9090; e-mail bernd.poetzsch{at}ukb.uni-bonn.de.

Background: Genetic analysis of platelet mRNA may facilitate the diagnosis of disorders affecting the megakaryocytic-platelet lineage. Its use, however, is limited by the exceptionally small yield of platelet mRNA and the risk of leukocyte contamination during platelet preparation.

Methods: We depleted platelet suspensions of leukocytes by filtration and used a PCR-based RNA amplification step [switching mechanism at the 5' end of RNA templates (SMART)]. We tested the reliability and precision of the RNA amplification procedure by use of real-time PCR to measure quantities of specific transcripts: von Willebrand factor (vWF), A-subunit of coagulation factor XIII (F13A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Microarray analysis was performed on platelet RNA with and without amplification.

Results: Microgram quantities of platelet-specific cDNAs were produced from as little as 50 ng of total platelet RNA or 40 mL of whole blood. At cycle numbers <16, amplification of all transcripts tested was exponential with slightly more efficient amplification of low-abundance transcripts. Expression profiling of 9850 genes gave identical results for 9815 genes (1576 positive/8239 negative). Eight transcripts failed to be amplified by the SMART procedure. Expression of vWF, F13A, and GAPDH transcripts showed only minor day-to-day variations in three healthy individuals.

Conclusion: The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.




The following articles in journals at HighWire Press have cited this article:


Home page
 BloodHome page
D. V. Gnatenko, P. L. Perrotta, and W. F. Bahou
Proteomic approaches to dissect platelet function: half the story
Blood, December 15, 2006; 108(13): 3983 - 3991.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
J. Wilhelm, J. P. Muyal, J. Best, G. Kwapiszewska, M. M. Stein, W. Seeger, R. M. Bohle, and L. Fink
Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments
Clin. Chem., June 1, 2006; 52(6): 1161 - 1167.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
W. Zhou, R. V. Abruzzese, I. Polejaeva, S. Davis, S. Davis, and W. Ji
Amplification of Nanogram Amounts of Total RNA by the SMART-Based PCR Method for High-Density Oligonucleotide Microarrays
Clin. Chem., December 1, 2005; 51(12): 2354 - 2356.
[Full Text] [PDF]

Home page
 Clin. Chem.Home page
N. Rolf, R. Knoefler, M. Suttorp, H. Kluter, and P. Bugert
Optimized Procedure for Platelet RNA Profiling from Blood Samples with Limited Platelet Numbers
Clin. Chem., June 1, 2005; 51(6): 1078 - 1080.
[Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2004 by the American Association for Clinical Chemistry.