"Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays

Kanno, Jun; Aisaki, Ken Ichi; Igarashi, Katsuhide; Nakatsu, Noriyuki; Ono, Atsushi; Kodama, Yukio; Nagao, Taku

doi:10.1186/1471-2164-7-64

Cited by 90 publications

(54 citation statements)

References 13 publications

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“…This approach minimizes the loss of signal intensity owing to biased purification procedures and allows for high through-put sample analysis using liquid handling robotics. Another advantage of this approach is that the lysates contain endogenous genomic DNA, which can be used to normalize the mRNA signals obtained in the RT-qPCR (15,16) without the need to analyse large panels of housekeeping genes (17). A potential disadvantage of this approach, on the other hand, is that most reagents used for preparing tissue lysates contain potent protein denaturants, high salt concentrations and chelating agents, which could inhibit or reduce the efficiency of the enzymatic reactions required for quantification (18).…”

Section: Resultsmentioning

confidence: 99%

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides

Boos

Kirk

Piccolotto

et al. 2013

Nucleic Acids Research

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Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

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Section: Resultsmentioning

confidence: 99%

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides

Boos

Kirk

Piccolotto

et al. 2013

Nucleic Acids Research

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“…Tissues were homogenized with a motor-driven micropestle. DNA quantitation was performed using Quanti-it™ PicoGreen® dsDNA Reagent (Invitrogen, Carlsbad, CA) as previously described by Kanno et al (2006).…”

Section: Methodsmentioning

confidence: 99%

Transgenerational Inheritance of Increased Fat Depot Size, Stem Cell Reprogramming, and Hepatic Steatosis Elicited by Prenatal Exposure to the Obesogen Tributyltin in Mice

Chamorro-García¹,

Sahu²,

Abbey³

et al. 2013

Environ Health Perspect

292

251

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Background: We have previously shown that exposure to tributyltin (TBT) modulates critical steps of adipogenesis through RXR/PPARγ and that prenatal TBT exposure predisposes multipotent mesenchymal stem cells (MSCs) to become adipocytes by epigenetic imprinting into the memory of the MSC compartment.Objective: We tested whether the effects of prenatal TBT exposure were heritable in F2 and F3 generations.Methods: We exposed C57BL/6J female mice (F0) to DMSO vehicle, the pharmaceutical obesogen rosiglitazone (ROSI), or TBT (5.42, 54.2, or 542 nM) throughout pregnancy via the drinking water. F1 offspring were bred to yield F2, and F2 mice were bred to produce F3. F1 animals were exposed in utero and F2 mice were potentially exposed as germ cells in the F1, but F3 animals were never exposed to the chemicals. We analyzed the effects of these exposures on fat depot weights, adipocyte number, adipocyte size, MSC programming, hepatic lipid accumulation, and hepatic gene expression in all three generations.Discussion: Prenatal TBT exposure increased most white adipose tissue (WAT) depot weights, adipocyte size, and adipocyte number, and reprogrammed MSCs toward the adipocyte lineage at the expense of bone in all three generations. Prenatal TBT exposure led to hepatic lipid accumulation and up-regulated hepatic expression of genes involved in lipid storage/transport, lipogenesis, and lipolysis in all three subsequent generations.Conclusions: Prenatal TBT exposure produced transgenerational effects on fat depots and induced a phenotype resembling nonalcoholic fatty liver disease through at least the F3 generation. These results show that early-life obesogen exposure can have lasting effects.

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“…A number of different biochemical approaches have been utilized to quantify mRNA copy number on a per cell basis such as serial analysis of gene expression (SAGE) (Velculescu et al ., 1999), quantitative PCR (qPCR) (Bengtsson et al ., 2008; Diercks et al ., 2009; Kanno et al ., 2006; Taniguchi et al ., 2009). Velculescu et al ., as early as 1999, published in Nature Genetics an analysis of the human transcriptome on a per cell basis using SAGE.…”

Section: Limitations Of Single-label Probes and Molecular Beaconsmentioning

confidence: 99%

“…This is likely due to the fact that these cells are the major insulin producers in the pancreas and therefore large quantities are produced. In another study by Kanno et al ., they focused on normalization methods for “per cell” data from qPCR and DNA microarray studies (Kanno et al ., 2006). Data over a large number of genes were collected, over 14,000 genes in the mouse genome.…”

Section: Limitations Of Single-label Probes and Molecular Beaconsmentioning

confidence: 99%

Probes for Intracellular RNA Imaging in Live Cells

Santangelo

Alonas

Jung

et al. 2012

Methods in Enzymology

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RNA localization, dynamics, and regulation are becoming increasingly important to our basic understanding of gene expression and RNA virus pathogenesis. An improved understanding of these processes will be necessary in order to identify new drug targets, as well as to create models of gene expression networks. Much of this new understanding will likely come from imaging studies of RNA, which can generate the spatiotemporal information necessary to characterize RNA within the cellular milieu. Ideally, this would be performed imaging native, nonengineered RNAs, but the approaches for performing these experiments are still evolving. In order for them to reach their potential, it is critical that they have characteristics that allow for the tracking of RNA throughout their life cycle. This chapter presents an overview of RNA imaging methodologies, and focuses on a single RNA sensitive method, employing exogenous probes, for imaging, native, nonengineered RNA in live cells.

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"Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays

Cited by 90 publications

References 13 publications

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides

Transgenerational Inheritance of Increased Fat Depot Size, Stem Cell Reprogramming, and Hepatic Steatosis Elicited by Prenatal Exposure to the Obesogen Tributyltin in Mice

Probes for Intracellular RNA Imaging in Live Cells

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