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Title: Accurate Gene and miRNA Quantification in Neuronal Differentiation
Authors: LIM QING 'EN
Keywords: microRNA, qPCR, normalization, differentiation, neuron, gene
Issue Date: 27-Sep-2011
Source: LIM QING 'EN (2011-09-27). Accurate Gene and miRNA Quantification in Neuronal Differentiation. ScholarBank@NUS Repository.
Abstract: Gene regulation is fundamental to cellular function. Neuronal differentiation is a critical process that involves precise regulation of many genes. microRNAs (miRNAs) have been found to be essential regulators of many biological processes including neuronal differentiation through their sequence-specific modulation of gene expression. Reverse transcription-quantitative PCR (RT-qPCR) is an established, sensitive and accurate platform for gene (both genomic and mRNA) quantitation. RT-qPCR has also been successfully applied to the quantification of miRNA. RT-qPCR or any other approach to gene quantification is dependent on valid comparisons between and/or within samples. Such comparisons most commonly involve comparison of the detected abundance gene(s) of interest against that of an endogenous reference gene. However, without a priori evidence of the stability of a reference gene, it is possible that interpretation of gene expression data could result in erroneous conclusions of gene regulation. It is therefore imperative to empirically determine the suitability of reference genes in any given experimental model. This work begins with the selection and use of endogenous reference genes for mRNA and miRNA studies in neuronal differentiation. Transcriptome-wide sampling supplemented by RT-qPCR gene quantification was used to empirically compare the stability of commonly used reference genes against novel reference genes. It emerged that mRNAs encoding ribosomal proteins but not popular reference genes such as GAPDH were stable reference genes in neuronal differentiation. To detect and quantify miRNAs, a RT-qPCR method previously used to quantify flaviviruses was adapted. This method was named modified stem-loop mediated reverse transcription-quantitative PCR (mSMRT-qPCR) and applied to the determination of stable miRNAs in neuronal quantification. We found that using a set of three miRNAs provided a more stable reference than the commonly used references snoU6 and 5S RNA. Finally, methods are described to adapt a qPCR buffer mixture for reverse transcription and to computationally aid designs of mSMRT-qPCR miRNA assays. The adapted reverse transcription buffer mixture is inherently compatible with downstream qPCR applications and benefits from the absence of PCR inhibitors such as dithiotreitol. To aid in the design and organization of a larger set of mSMRT-qPCR primer designs, a design platform was implemented using Microsoft Excel. miRNA assays designed using this platform were successfully used to detect miRNAs from both isolated RNA and whole cell lysate.
Appears in Collections:Master's Theses (Open)

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