Neuroprotective role of Sirt1 in mammalian models of Huntington's disease through activation of multiple Sirt1 targets.

  • Dahl T
  • Holm S
  • Aukrust P
 et al. 
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Abstract

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

Author-supplied keywords

  • Animal
  • Animals
  • Brain
  • Brain-Derived Neurotrophic Factor
  • Carcinoma
  • Cell Differentiation
  • Cell Differentiation: physiology
  • Cell Differentiation: radiation effects
  • DNA Primers
  • DNA Primers: genetics
  • Deacetylation
  • Disease Models
  • Dopamine and cAMP-Regulated Phosphoprotein 32
  • Forkhead Transcription Factors
  • Gene Expression Regulation
  • Humans
  • Huntington Disease
  • Huntington's disease
  • Keratinocytes
  • Keratinocytes: metabolism
  • Keratinocytes: physiology
  • Lysine deacetylases
  • Mice
  • MicroRNAs
  • MicroRNAs: metabolism
  • Microarray Analysis
  • Neoplastic
  • Neoplastic: physiology
  • Nerve Tissue Proteins
  • Neurons
  • Neuroprotection
  • Nicotinamide mononucleotide
  • Nicotinamide phosphoribosyltransferase
  • Nicotinamide riboside
  • Notch1
  • Notch1: metabolism
  • Nuclear Proteins
  • Poly-ADP-ribose polymerases
  • Rats
  • Real-Time Polymerase Chain Reaction
  • Receptor
  • SIRT1
  • Signal Transduction
  • Sirtuin 1
  • Sirtuins
  • Sirtuins: metabolism
  • Squamous Cell
  • Squamous Cell: metabolism
  • Therapeutic targets
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Protein p53: metabolism
  • Ultraviolet Rays
  • aging
  • atherosclerosis
  • cancer
  • cardiovascular
  • chromatin
  • cognition
  • cytokine
  • deacetylase
  • intracellular enzyme
  • metabolic disorders
  • metabolism
  • metastasis
  • miR-34
  • miR-34a
  • miR-34b/c
  • neurodegeneration
  • p53
  • sirt1
  • trkB
  • tumor suppression

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Authors

  • Tuva B. Dahl

  • Sverre Holm

  • Pål Aukrust

  • Bente Halvorsen

  • Marcia C Haigis

  • David a Sinclair

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