High quality RNA extraction of the mammalian cochlea for qRT-PCR and transcriptome analyses

Hear Res. 2015 Jul;325:42-8. doi: 10.1016/j.heares.2015.03.008. Epub 2015 Mar 27.

Abstract

Molecular investigations of the hearing organ, the cochlea, have been hampered due to the difficulty of isolating pure RNA and in quantities sufficient enough for quantitative real-time RT-PCR or microarray analysis. The complex architecture of the cochlea, the presence of liquids, bone and cartilage tissue, are a major hurdle in obtaining contamination-free RNA to a level that does not affect downstream applications. Here, we present a protocol to extract RNA from the mouse cochlea, with yields and quality suitable for real-time RT-PCR or Affymetrix labeling. In contrast to current methods, such as TRIZOL or column-based extraction, this protocol combines the two and, within 4 h, yields a 2 μg of total RNA from a single pair of adult mouse cochleae. This protocol allows the isolation of RNA molecules from the mammalian cochlea providing access to whole-transcript expression analyses.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone and Bones / metabolism
  • Cartilage / metabolism
  • Chloroform / chemistry
  • Circadian Rhythm
  • Cochlea / metabolism*
  • DNA / chemistry
  • Edetic Acid / chemistry
  • Gene Expression Profiling
  • Guanidines / chemistry
  • Mice
  • Microfluidics
  • Oligonucleotide Array Sequence Analysis*
  • Phenols / chemistry
  • RNA / chemistry*
  • Real-Time Polymerase Chain Reaction
  • Spectrophotometry

Substances

  • Guanidines
  • Phenols
  • trizol
  • RNA
  • Chloroform
  • DNA
  • Edetic Acid