Open AccessFreezing fecal samples prior to DNA extraction affects the F irmicutes to B acteroidetes ratio determined by downstream quantitative PCR analysis
Author(s) -
Bahl Martin Iain,
Bergström Anders,
Licht Tine Rask
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2012.02523.x
Subject(s) - bacteroidetes , firmicutes , biology , dna extraction , feces , metagenomics , 16s ribosomal rna , microbiology and biotechnology , polymerase chain reaction , real time polymerase chain reaction , extraction (chemistry) , dna , food science , bacteria , chromatography , gene , chemistry , genetics
Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16 S r RNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the F irmicutes to B acteroidetes 16 S r RNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by q PCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined F irmicutes to B acteroidetes ratio, which is a frequently used biomarker in gut microbiology.