Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures

Mol Neurobiol. 2016 Apr;53(3):1540-1550. doi: 10.1007/s12035-015-9114-5. Epub 2015 Feb 7.

Abstract

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

Keywords: Ageing; Normalization; Normfinder; RT-qPCR; geNorm.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aging / genetics*
  • Algorithms
  • Animals
  • Brain Chemistry / genetics*
  • Cerebellum / metabolism
  • Cerebral Cortex / growth & development
  • Cerebral Cortex / metabolism
  • Corpus Striatum / growth & development
  • Corpus Striatum / metabolism
  • Gene Expression Profiling*
  • Hippocampus / metabolism
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Nerve Tissue Proteins / biosynthesis*
  • Nerve Tissue Proteins / genetics
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Nerve Tissue Proteins