Molecular investigations on a chimeric strain of Staphylococcus aureus sequence type 80

An Eritrean patient was admitted with suspected tuberculous cervical lymphadenitis. While no mycobacteria were detected in pus from this process, culture yielded PVL-positive, methicillin-susceptible Staphylococcus aureus. Microarray hybridisation assigned the isolate to clonal complex (CC) 80 but revealed unusual features, including the presence of the ORF-CM14 enterotoxin homologue and of an ACME-III element as well as the absence of etD and edinB. The isolate was subjected to both, Illumina and Nanopore sequencing allowing characterisation of deviating regions within the strain’s genome. Atypical features of this strain were attributable to the presence of two genomic regions that originated from other S. aureus lineages and that comprised, respectively, 3% and 1.4% of the genome. One deviating region extended from walJ to sirB. It comprised ORF-CM14 and the ACME-III element. A homologous, but larger fragment was also found in an atypical S. aureus CC1/ST567 strain whose lineage might have served as donor of this genomic region. This region itself is a chimera comprising fragments from CC1 as well as fragments of unknown origin. The other region of another 3% of the genome comprised the region from htsB to ecfA2. It was very similar to CC1 sequences. This suggests either an incorporation of CC1 DNA into the study strain, or it might alternatively suggest a recombination event affecting “canonical” CC80. As the study strain bears witness of several recombination events, such complex and large-scale events cannot be rare and exceptional, despite a mainly clonal nature of S. aureus. Although the exact mechanism is not yet clear, chimerism seems to be an additional pathway in the evolution of S. aureus, possibly being responsible for the transmission also of virulence and resistance factors. An organism that can shuffle, swap or exchange major parts of its genome by a yet unknown mechanism would have an evolutionary advantage compared to a strictly clonal organism.

Comparability of Microarray Data between Amplified and Non Amplified RNA in Colorectal Carcinoma

Microarray analysis reaches increasing popularity during the investigation of prognostic gene clusters in oncology. The standardisation of technical procedures will be essential to compare various datasets produced by different research groups. In several projects the amount of available tissue is limited. In such cases the preamplification of RNA might be necessary prior to microarray hybridisation. To evaluate the comparability of microarray results generated either by amplified or non amplified RNA we isolated RNA from colorectal cancer samples (stage UICC IV) following tumour tissue enrichment by macroscopic manual dissection (CMD). One part of the RNA was directly labelled and hybridised to GeneChips (HG-U133A, Affymetrix), the other part of the RNA was amplified according to the “Eberwine” protocol and was then hybridised to the microarrays. During unsupervised hierarchical clustering the samples were divided in groups regarding the RNA pre-treatment and 5.726 differentially expressed genes were identified. Using independent microarray data of 31 amplified vs. 24 non amplified RNA samples from colon carcinomas (stage UICC III) in a set of 50 predictive genes we validated the amplification bias. In conclusion microarray data resulting from different pre-processing regarding RNA pre-amplification can not be compared within one analysis.

A guide to issues in microarray analysis: application to endometrial biology

Within the last decade, the development of DNA microarray technology has enabled the simultaneous measurement of thousands of gene transcripts in a biological sample. Conducting a microarray study is a multi-step process; starting with a well-defined biological question, moving through experimental design, target RNA preparation, microarray hybridisation, image acquisition and data analysis – finishing with a biological interpretation requiring further study. Advances continue to be made in microarray quality and methods of statistical analysis, improving the reliability and therefore appeal of microarray analysis for a wide range of biological questions. The purpose of this review is to provide both an introduction to microarray methodology, as well as a practical guide to the use of microarrays for gene expression analysis, using endometrial biology as an example of the applications of this technology. While recommendations are based on previous experience in our laboratory, this review also summarises the methods currently considered to be best practice in the field.