Rneasy dsp ffpe kit

Total RNA was extracted from an at least 6 µm FFPE tissue section with a tumor content ≥ 20%, using the RNeasy^® DSP FFPE Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instruction. The RNeasy DSP FFPE Kit was validated by APIS as an RNA extraction method to be used in conjunction with the APIS BC Subtyping Kit. For each specimen, RNA within the eluate was fluorometrically quantified using Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), normalized to 2.5 ng/µL and stored at −80 °C until use.

MicroRNA extraction was performed from paraffin tumor tissue sections (RNeasy DSP FFPE Kit, Qiagen) and the RNA amount was determined using a NanoDrop spectrophotometer. Differently, miRNAs obtained from serum patients (200 μl) were isolated by the Qiagen miRNeasy kit with further modifications for biofluids applications. Syn-cel-miR-39 spike in synthetic RNA (Qiagen) was added to monitor extraction efficiency. Afterward, on both tissue sections and sera samples the reverse transcription was performed using the “MiRCURY LNA Reverse Transcription Kit” (QIAGEN) in ThermoMixer 5436 (Eppendorf, Italy) according to the following protocol: 42°C for 60 seconds, 95°C for 5 minutes, 4°C forever [33 (link)].
Selected miRNA levels such as hsa-miR-101, hsa-miR-126-3p, hsa-miR-486, and hsa-Let-7g were quantified by relative quantification using Qiagen LNA-based SYBR green detection method (miRCURY LNA miRNA PCR assay- Qiagen). Briefly, 3 μl of cDNA was used on the Applied Biosystems 7900HT machine, adding the relevant ROX concentration to the qPCR mix [33 (link)]. The relative miRNA expression was calculated using hsa-miR-16 as endogenous control with the 2-ΔCt method for cancer tissues and miR-16/miR-39 for sera [33 (link)].

Total RNA was isolated with the RNeasy DSP FFPE Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and 260/280 absorbance ratio (A260/280) was measured with the Infinite M200 spectrophotometer (TECAN) and quality control of extracted RNA assessed with RT-qPCR for 18S rRNA. RNA from ACC (h-TERT) cells was kindly gifted by Prof. Sandra Sigala (University of Brescia).
To generate gene expression profiles, total RNA was converted to cRNA and then to cDNA, and hybridized, according to the manufacturer’s guidelines, on the Human Clariom S GeneChip (ThermoFisher Scientific), optimized for FFPE samples and able to accurately measure gene-level expression from > 20,000 well-annotated genes. The Scanner 3000 7G (ThermoFisher Scientific) was used in conjunction with GeneChip Operation Software (ThermoFisher Scientific) to generate one CEL file per hybridized cDNA.