A multiplex branched DNA assay for parallel quantitative gene expression profiling

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L.; McMaster, Gary; Witney, Frank; Luo, Yuling

doi:10.1016/j.ab.2006.02.013

Cited by 100 publications

(91 citation statements)

References 22 publications

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“…1) [17,27]. It has been routinely used as an alternative for detection of mRNA expression and siRNA efficacy in cell culture assays for the past decade [15,[17][18][19][20][21]. Here, we evaluated the merit of using the bDNA assay for detecting mRNA expression directly from tissue biopsies, by using the automatic TissueLyser II (Qiagen) for tissue homogenization, followed with direct detection of mRNA levels by bDNA (see Materials and Methods for experimental details).…”

Section: Resultsmentioning

confidence: 99%

“…The QuantiGene Ò branched DNA (bDNA) assay was developed to circumvent the pitfalls associated with qRT-PCR [15,16]. Unlike qRT-PCR, this bDNA assay relies on amplification of the signal, not the cDNA of interest.…”

mentioning

confidence: 99%

“…This method utilizes a plate-based, luminescent assay to directly assess the amount of mRNA in 96 samples simultaneously, without the need to isolate RNA or include a standard curve [17]. It has been used routinely for diagnostics, and to a lesser extent, to assess the efficacy of oligonucleotides in vivo [15,[17][18][19][20][21]. A previous report compared qRT-PCR to the QuantiGene assay and determined that QuantiGene has a better linear range and relative accuracy versus qRT-PCR [22].…”

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confidence: 99%

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A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

Coles

Osborn

Alterman

et al. 2016

Nucleic Acid Therapeutics

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Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene Ò branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra-and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

Coles

Osborn

Alterman

et al. 2016

Nucleic Acid Therapeutics

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“…The multiplex bDNA assay for blood is similar to a previously published multiplex assay procedure (24 ). A panel of oligonucleotide capture probes, each with a unique sequence of 15 bases, were synthesized with 5Ј-amino linker (BioSearch), and each was covalently linked to carboxylated fluorescently encoded beads (Luminex) according to the recommended conjugation procedure.…”

Section: Multiplex Assaymentioning

confidence: 99%

“…To simultaneously detect several distinct mRNAs in whole-blood lysate, we developed a multiplexed assay based on a Luminex bead-based multiplex platform (24,28 ) with modifications for higher stringency (see Materials and Methods). Target-specific hybridization occurs on the surface of different internally color-coded microbeads.…”

Section: Assay Performancementioning

confidence: 99%

Sensitive and Quantitative Measurement of Gene Expression Directly from a Small Amount of Whole Blood

Zheng¹,

Luo²,

McMaster³

2006

Clinical Chemistry

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Background: Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format.

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Real‐Time Quantitative RT‐PCR for mRNA Profiling

Bustin

Nolan²

2010

Genomics

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The real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) [1] is characterized by its specificity, sensitivity, simplicity and speed. These attributes have made it the method of choice for the detection and quantification of RNA [2, 3], and have effected its extensive use in biotechnology [4], microbiology [5], virology [6] and molecular medicine [7] applications. The assay involves a conventional reverse transcription (RT) procedure, followed by the qPCR step, which makes use of fluorescent reporter molecules to combine the amplification and detection components of the PCR in a single tube format [8,9]. An increase in fluorescent signal is proportional to the amount of DNA produced during each PCR cycle and produces a characteristic threshold cycle (C t ) or crossing point (C p ) for every reaction. The C t or C p is defined as the PCR cycle at which the signal first rises above background fluorescence. The more target there is in the starting material, the earlier the instrument can detect the fluorescence and the lower the C t . This correlation between fluorescence and amount of amplified product permits accurate quantification of target molecules over a wide dynamic range and the homogeneous format significantly reduces hands-on time and the risk of contamination [10].The consistency and reliability of RT-qPCR assays depend on the proper execution of a number of steps, in particular those relating to sample selection, template quality, assay design and data analysis [11], as well as the correct application of statistical models and methods for data analysis [12]. If correct procedure is adhered to, then results are generally robust, reproducible and accurate. Specifically, the reliability of the RT-qPCR assay is Genomics: Essential M ethodsEdite d by Mike S ta r ke y a nd R a mna th Ela swa r a pu

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A multiplex branched DNA assay for parallel quantitative gene expression profiling

Cited by 100 publications

References 22 publications

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

Sensitive and Quantitative Measurement of Gene Expression Directly from a Small Amount of Whole Blood

Real‐Time Quantitative RT‐PCR for mRNA Profiling

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