VEGF-A mRNA measurement in meningiomas using a new simplified approach: branched DNA and chemiluminescence

Abstract

For decades, the preferred and almost sole method for measurement of gene expression has been RT-qPCR. The method is robust, inexpensive, and well-studied; however, PCR is also quite laborious and vulnerable to contamination. As part of an investigation of VEGF-A gene expression in meningiomas, an alternative and less laborious method for gene expression analysis based on branched DNA hybridization and chemiluminescence (Lumistar) was tested. Albeit the two methods differ, in principle, cellular mRNA-concentration is measured with both. Because they both determine gene expression via the measurement of mRNA-concentration, they were expected to be comparable. The aim of the present study was to compare Lumistar to the traditional RT-qPCR approach in a routine laboratory setting, where there is emphasis on rapid analysis response. Meningioma (n = 10) and control brain tissue (n = 5) samples were collected and VEGF-A and GAPDH mRNA were quantified using both RT-qPCR and Lumistar. Furthermore, two dilution series of two of the meningioma samples were prepared in order to make quantitative analyses. Both Lumistar and RT-qPCR-results were found to follow concentration dependent linear paths when diluted (p < 0.0001 and p < 0.01). Finally, Lumistar and RT-qPCR analyses were performed with the inclusion of a reference gene (GAPDH), where similar results were obtained with the two methods (R2 = 0.48; p = 0.01). It is intriguing that in spite of the vast difference in handling and assay principles, gene expression results are similar. The preferred method depends on the variability of the samples, budget, and time. Lumistar was less time consuming, while RT-qPCR was less expensive and best suited for data sets with large sample variability.

Role of Bevacizumab in High-grade Meningiomas

“…The importance of deregulated cell signaling pathways as drivers of neoplastic transformation is increasingly getting attention. Several studies have suggested a critical role of VEGF in meningioma pathogenesis, as its expression correlates with tumor grade, peritumoral edema, and necrosis [22,23]. For this reason, in the last few years, antiangiogenetic factors have been used, not only for malignant glial brain tumors but also for meningiomas not responsive to standard treatments [24].…”

DNA Functional Materials Assembled from Branched DNA: Design, Synthesis, and Applications

“…371,402 In addition, this method could obtain the measurement results of mRNA concentration similar to traditional real-time quantitative PCR (RT-qPCR) approach, and was a sensitive, accurate, less-laborious, and automated tool alternative to the RT-qPCR. 403 The PCR-free branched DNA assay therefore was conducive to the quantitative detection of target transcripts in mRNA population for early disease markers. 404 4.1.2.…”

“…It is noteworthy that the technique did not require the participation of enzymes and rigorous experimental conditions. By virtue of the technology, Liu et al introduced SA-coated magnetic beads as fixed substrates and Ru­(bpy) 3 2+ -labeled probes in replace of traditional alkaline phosphatase labels, consequently achieving the ultrasensitive electro­chemiluminescence detection of target DNA in pathogenic bacteria, Staphylococcus aureus . , In addition, this method could obtain the measurement results of mRNA concentration similar to traditional real-time quantitative PCR (RT-qPCR) approach, and was a sensitive, accurate, less-laborious, and automated tool alternative to the RT-qPCR . The PCR-free branched DNA assay therefore was conducive to the quantitative detection of target transcripts in mRNA population for early disease markers …”