Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection

Abstract

Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.

Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

“…Furthermore, since leukocytes contain approximately 12 500-fold more mRNA than platelets, residually contaminated leukocytes could affect mRNA levels in platelets and thus they might be overestimated in platelets. 39 Inclusion bodies are present in granulocytes but not in lymphocytes in patients with MYH9 disorders. 1,2 Accordingly, whether mutant NMMHC-IIA is expressed in lymphocytes has remained obscure.…”

TLR7 Ligands Induce Higher IFN-α Production in Females

“…was performed by our group as described in detail previously (41,42). One milliliter of the purified pDCs in suspension was spun down for 8 min at 1200 rpm on glass slides (SuperFrost Plus; Menzel-Glaeser) using the Cytospin 2 centrifuge (Thermo Shandon).…”

“…Reagents, as well as primers, were applied as described previously (41). Eleven microliters of cDNA was subjected to PCR amplification.…”

Biotecnologia Aplicada à Agro&Indústria - Vol. 4

“…Estudo de Fink (2003) 40 demonstrou que essa técnica aliada à manipulação assistida por laser mostrou-se eficiente na acurácia para detectar células sanguíneas, as quais poderiam ser plaquetas ou não. Para a contagem de células sanguíneas, os analisadores automatizados passaram por avanços, os quais determinam hoje com maior eficiência a diferenciação dessas célu-las, a partir da propriedade de dispersão da luz, a absorbância.…”