QuantiGene Singleplex Gene Expression Assays

QuantiGene Singleplex Gene Expression Assays help provide a streamlined approach to RNA quantification, eliminating the need for reverse transcription or PCR. This assay offers exceptional accuracy and reliability, making it excellent for a wide range of gene expression studies. With its robust performance and simplified workflow, the QuantiGene Singleplex Assay is an important tool for researchers aiming to advance their molecular biology research.

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QuantiGene Singleplex assays help provide a fast and high-throughput solution for gene expression quantitation, allowing measurement of a gene of interest in a single well of a 96- or 384-well plate. This hybridization-based assay incorporates branched DNA (bDNA) technology, using signal amplification rather than target amplification for direct measurement of RNA transcripts. With its robust performance and high sensitivity, the QuantiGene Singleplex Assay simplifies workflows with an easy-to-use, ELISA-like process that eliminates the need for RNA purification, reverse transcription, or qPCR (Figure 1). Well suited for a variety of research applications, including biomarker discovery, validation, and molecular diagnostics, this innovative assay enhances research capabilities by offering remarkable accuracy and efficiency in gene expression studies.

QuantiGene assays utilize branched DNA (bDNA) technology, which utilizes sequential nucleic acid hybridization for a distinct approach to RNA and DNA quantification by amplifying a reporter signal rather than the template. First, cells are lysed or tissue samples are homogenized to release the target RNA. Second, the oligonucleotide probe set is incubated with the target RNA and capture plate overnight. During this incubation, the probes cooperatively hybridize to the target and capture probes bound to the plate, capturing the target RNA. Third, signal amplification is performed via sequential hybridization of the bDNA preamplifier, amplifier, and label-probe molecules to the target. Addition of a chemiluminescent substrate generates a luminescence signal directly proportional to the amount of target mRNA present in the sample (Figure 2). The resulting signal is then read on a luminometer, such as the Varioskan ALF Multimode Microplate Reader.

Figure 2. How QuantiGene Singleplex assays work. Samples are prepared to get lysate with target RNA. The target of interest is then hybridized onto the wells of the microplate through a series of steps. Signal amplification and detection is then performed, with the signal read on a luminometer.

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• Direct from lysate quantitation using signal amplification—no RNA or DNA purification, reverse transcription, or PCR amplification needed
• No bias avoids the enzymatic bias inherent to reverse transcription and target amplification
• Precisely quantify subtle differences in gene expression as small as 10% (Figure 3)
• Simple workflow features lyse-and-go sample preparation, easily automatable ELISA-like protocol, and readout using any standard luminometer
• Analyze any sample type: cultured cells, whole or dried blood, plants, bacteria, tissue homogenates, and difficult samples such as formalin-fixed paraffin-embedded (FFPE) (Figure 4)
• Exceptional specificity delivers greater precision than other common technologies, and distinguishes between closely related genes due to probe design with six capture points along the target mRNA of interest
• Assay flexibility allows for easily automated use in routine compound screening
• Expand your research—probes are also available for DNA copy number analysis and microRNA expression
• Automation—ELISA-like workflow allows for easy adoption of high-throughput screening

QuantiGene Singleplex assays are exceptional tools for a wide range of applications in molecular biology and research.

• Compound screening of drug candidates—evaluate the efficacy and toxicity of potential therapeutics
• Biomarker verification—precisely quantify and verify biomarkers in clinical and preclinical studies
• siRNA knockdown efficiency—accurately assess gene suppression in gene silencing studies
• Prospective and retrospective analysis of clinical samples—obtain reliable data for longitudinal studies and historical sample evaluation
• miRNA profiling—gain insights into gene regulation mechanisms
• Microarray verification—help ensure the accuracy of high-throughput gene expression data
• Predictive toxicology—foresee and mitigate adverse effects of new compounds
• Detection of translocations and fusion genes—identify genetic abnormalities associated with various diseases
• DNA assay—used for copy number determination in transgenic animals, plant zygocity testing

Read more about QuantiGene assays and how they are used in the following various immunoassay research areas:

• Cell signaling
• Inflammation and cytokine storm research
• Neurobiology and neurodegenerative disorders

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To demonstrate the accuracy of the QuantiGene Singleplex Gene Expression Assay, measurements of twenty transcripts from two reference RNA samples were made and compared to TaqMan data. Reference RNA samples were human brain total RNA and universal human reference RNA [2]. As shown in Figure 5, excellent correlations in fold-change of RNA levels in the reference samples were seen between QuantiGene SinglePlex and TaqMan assays (R² = 0.965).

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In drug discovery, time is money. Fast-track your discovery using the QuantiGene Singleplex High Throughput (HT) Assay, which enables gene expression-based high-throughput screening of thousands of compounds every day. The assay's ELISA-like workflow is easy to automate and does not require RNA purification or extensive assay optimization. The extremely robust QuantiGene Singleplex HT Assay is hybridization-based and uses no enzymes; instead, it relies on branched DNA technology for signal amplification and luminescence for signal readout in a convenient 384-well plate.

Distinct benefits:

• Quantitate a variety of samples—including cultured cells, whole blood, dried blood spots, fresh/frozen or FFPE samples, bacteria and purified RNA/DNA
• High throughput—simple ELISA-like workflow is easily automated with liquid handling and dispensing platforms, with no RNA purification needed
• Robust and reproducible—high Z'-factors are typically attained with the QuantiGene Singleplex HT Assay, giving you more confidence in your hits
• Easy to use—no optimization of probe sets is required, sample preparation is minimal, and 384-well plate format allows for direct transfer of cells cultured in 384-well plates

High throughput QuantiGene assays have been used by researchers such as Gozgit et al., who investigated the modulation of CYP1B1 levels by PARP7. In the Cancer Cell article, they found that PARP7 is a cancer-specific brake in cytosolic nucleic acid sensing. HARA cells were lysed and then used with the assay kit QS0384 and the corresponding PARP7 probe set [3].

1. Beer, David G et al. Gene-expression profiles predict survival of patients with lung adenocarcinoma.Nature medicine vol. 8,8 (2002): 816–824. doi:10.1038/nm733.
2. Canales RD, Luo Y, Willey JC, et al. Evaluation of DNA microarray results with quantitative gene expression platforms. Nat Biotechnol. 2006;24(9):1115–1122. doi:10.1038/nbt1236.
3. Gozgit JM, Vasbinder MM, Abo RP, et al. PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell. 2021;39(9):1214–1226.e10. doi:10.1016/j.ccell.2021.06.018.

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