5. Molecular profiling of bacterial DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue: a comparative study

Abstract

There is increasing use of FFPE tissues for molecular profiling. We sought to determine the effects of fixation on our ability to detect bacterial DNA in tissues. Aim To investigate the effect of FFPE, and two de-paraffinisation methods on DNA yields, bacterial DNA amplification and molecular fingerprinting. Methods Intestinal tissue and corresponding gut microflora were collected from mice and chickens. Triplicate samples from 14 animals were either snap frozen at -80°C (FS); or fixed in 10% formalin prior to processing. The FFPE samples were de-paraffinised using either the traditional method (TM) or a microwave method (MW). DNA was extracted from all tissues using a modified commercial kit protocol. DNA yields were determined, and amplification of bacterial 16S rDNA fragments compared. Denaturing gradient gel electrophoresis (DGGE) was used to generate ‘fingerprints’ of PCR amplicons to obtain an estimate of number of species of gut microbiota present. Results DNA yields from FFPE-TM and -MW tissues were 11% and 35% respectively of those from FS. 16S PCR products were obtained from 93% of FS, 7% of FFPE-TM and 57% of FFPE-MW samples. DGGE profiles showed FS yielded PCR fingerprints with the most bands (avg 22 bands) followed by FFPE-MW (avg 3 bands). Conclusion Compared to FS, FFPE tissue yields less bacterial DNA, and is less likely to enable the generation of PCR amplicons or DGGE bands for molecular profiling. The MW method yielded twice the DNA of TM. The amplification success of the 260 bp 16S rDNA fragment is poor when DNA has been extracted from FFPE, regardless of de-paraffinisation method.