Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
Methods Mol Biol. 2025;2857:89-98. doi: 10.1007/978-1-0716-4128-6_8.
QuantiGene™ 2.0 technique could be used to investigate the gene expression signature of the immune system senescence and thus to understand the molecular mechanism involved in the defects of the immune response during aging.QuantiGene™ 2.0 technique is a multiplex platform allowing the simultaneous analysis of several target RNA molecules (up to 80) present in a single sample. QuantiGene Assays use an accurate method for multiplexed or for single gene expression quantitation. QuantiGene 2.0 uses magnetic beads which are dyed internally with two fluorescence dyes, exhibiting a unique spectral signal and providing specificity and multiplexing capability of the technique. QuantiGene Assays incorporate branched-DNA technology for gene expression profiling.Branched-DNA system is responsible for the high sensitivity of the system. In fact, it permits to detect low levels of mRNA molecules. This branched-DNA system allows for the direct measurement of RNA transcripts by using signal amplification rather than target amplification. The assay protocol is spread over 2 days. First, immune cells are lysed to release the target RNA, which is incubated with oligonucleotide probe set targeted with beads capable to hybridize with the target RNA. Signal amplification is performed by sequential hybridization of the branched-DNA pre-amplifier, amplifier, and label probe molecules. The last step involves the incubation with Streptavidin-conjugated R-phycoerythrin. The fluorescent reporter generates a signal directly proportional to the levels of RNA molecules present in the cells. Luminex instrument evaluates the median intensity of fluorescence, which is proportional to the number of RNA target molecules present in the cells.
QuantiGene™ 2.0 技术可用于研究免疫系统衰老的基因表达特征，从而了解衰老过程中免疫反应缺陷所涉及的分子机制。QuantiGene™ 2.0 技术是一种多重平台，允许同时分析单个样本中存在的多个靶 RNA 分子（多达 80 个）。QuantiGene 检测使用一种准确的方法进行多重或单个基因表达定量。QuantiGene 2.0 使用内部染有两种荧光染料的磁性珠，呈现独特的光谱信号，并提供技术的特异性和多重能力。QuantiGene 检测采用分支 DNA 技术进行基因表达谱分析。分支 DNA 系统是该系统高灵敏度的原因。事实上，它允许检测低水平的 mRNA 分子。该分支 DNA 系统通过信号放大而不是目标物放大来直接测量 RNA 转录物。检测方案需要 2 天时间。首先，免疫细胞被裂解以释放靶 RNA，然后将其与寡核苷酸探针组孵育，探针组与能够与靶 RNA 杂交的珠结合。信号放大通过分支 DNA 预扩增子、放大器和标记探针分子的顺序杂交来进行。最后一步涉及与链霉亲和素偶联的藻红蛋白孵育。荧光报告物产生的信号与细胞中存在的 RNA 分子的水平直接成正比。Luminex 仪器评估荧光强度的中位数，这与细胞中存在的 RNA 靶分子的数量成正比。
