Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer
| Autor: | Prokopis Konstanti, Suk Yee Lam, Maikel P. Peppelenbosch, Gwenny M. Fuhler, Thijmen Visseren, Clara Belzer, Athanasia Ioannou, Michail Doukas |
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| Přispěvatelé: | Gastroenterology & Hepatology, Pathology |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
DNA
Bacterial Microbiology (medical) Faecalibacterium prausnitzii medicine.disease_cause Microbiology Colorectal neoplasms Specimen Handling Formalin-fixed paraffin embedded SDG 3 - Good Health and Well-being Microbiologie Formaldehyde RNA Ribosomal 16S medicine Humans MolEco Escherichia coli Gastrointestinal microbiome VLAG Low biomass Paraffin Embedding Bacteria biology Research Sequence Analysis DNA biology.organism_classification 16S ribosomal RNA DNA contamination Molecular biology DNA extraction QR1-502 UniFrac Real-time polymerase chain reaction DNA Contamination Nested polymerase chain reaction High-throughput nucleotide sequencing |
| Zdroj: | BMC Microbiology 21 (2021) BMC Microbiology, 21 BMC Microbiology BMC Microbiology, 21(1):297. BioMed Central Ltd. BMC Microbiology, Vol 21, Iss 1, Pp 1-11 (2021) |
| ISSN: | 1471-2180 |
| Popis: | Background Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants. |
| Databáze: | OpenAIRE |
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