Quantitative Real-Time PCR for Equine Cytokine mRNA in Nondecalcified Bone Tissue Embedded in Methyl Methacrylate

Abstract.

Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and embedded in a hydrophobic acrylic resin. Total RNA was extracted from defined regions of single 50 μm sawed sections. These RNA preparations are suitable for quantitative PCR analysis of mRNA of different cytokines. In addition, the universally expressed housekeeping GAPDH mRNA proved to be useful as an amplification control and to correct for the degree of RNA degradation, which may vary considerably among samples. Reverse transcribed mRNA was amplified and quantitated in Real-Time PCR using a fluorescein labeled internal TaqMan® probe.