Rotor gene q mdx instrument

All the retrieved hematoxylin and eosin (H&E) stained sections for the cohort cases were reviewed separately by two experienced histopathologists at the Endocrine Pathology Department within our institution. Both pathologists reviewed and confirmed the diagnoses of papillary thyroid carcinoma.
The most appropriate slide for BRAF molecular testing was determined based on the percentage of primary thyroid tumour and lymph node metastases if present. A cut-off point of 10% was deemed the minimal accepted tumour percentage on the selected slides. Five sections of approximately 5–10 µm thickness were sectioned from the formalin-fixed paraffin-embedded (FFPE) tumour tissue corresponding to the selected slides. Sectioned tissues were collected in Eppendorf tubes and labelled appropriately. The DNA was extracted and purified using the QIAamp® DNA Mini Kit (Qiagen). Samples were assessed for DNA concentration and purity using the NanoDrop® ND-1000 spectrophotometer. BRAF mutation testing was performed using therascreen® BRAF RGQ PCR Kit on the QIAGEN Rotor-Gene Q MDx instrument, designed to detect five somatic mutations in the BRAF gene including V600E, V600E complex (V600Ec), V600D, V600K, and V600R.

The expression of a selection of genes that were differentially expressed in the RNA-seq analysis was quantified by RT-qPCR. Total RNA from G. sulfurreducens biofilms was obtained as described above. cDNA was synthesized using the Revert Aid First Strand DNA Synthesis kit (Thermo Scientific) and the specific reverse oligonucleotides listed in S1 Table. RT-qPCR was then performed using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) on a Rotor-Gene® Q MDx instrument (Qiagen). The relative expression of the target genes was calculated with the Rotor-Gene Q Series Software using the 2^-ΔΔCT method. The expression of the gsu2822 gene was used as an internal control. All reactions were performed in triplicate and their average values were calculated.