Purelink ffpe total rna isolation kit

Total RNA was purified from three pooled sections of the same tissue block using the Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) as described previously [41 (link)]. RT-PCR was carried out using the QuantiTect Virus Kit (Qiagen, Hilden, Germany) in a total volume of 25 μL containing 5 μL of 5xQuantiTect Virus Mastermix, 0.25 μL of 100xQuantiTect Virus RT Mix, 0.4 μM of each oligonucleotide, and 1 μL RNA as described previously [42 (link)]. The HPV type-specific E6^*I mRNA assay developed for 20 HR- or pHR-HPV types was applied for the detection of viral transcripts. The assay amplifies a 65–75 base pair amplicon of HPV and an 81 base pair amplicon of ubiquitin C (ubC) cDNA.
The HPV RNA analysis was performed at the German Cancer Research Center laboratories in Heidelberg, Germany.

Total RNA was extracted using the commercially available illustraRNAspin Mini Isolation Kit (GE Healthcare, Italy), according to manufacturer's instructions. Tissue RNAs was extracted using the commercially available PureLink FFPE Total RNA Isolation Kit (Invitrogen cat.number 45-7015). Human colorectal cancer specimens (n = 22) were collected from the pathology archives of the Human Pathology Section, Ospedali Riuniti Villa Sofia-Cervello (Palermo) in accordance with the Declaration of Helsinki and with the policy of the Institute. RNA was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, USA). Real-time PCR was performed in duplicates for each data point, and the oligonucleotides used are described in Table 1.
Changes in the target mRNA content relative to housekeeping gene (β-actin) were determined with the ΔΔct Method. For miRNA expression, 250 ng of RNA was reverse transcripted according to manufacturer's instructions (cat.number 4366596, TaqManMicroRNA Reverse Transcription, Applied Biosystem). Taqman probes were used to analyse: miR-675-5p (cat.number 4440887, Applied Biosystem), miR-675-3p (cat.number 4427975, Applied Biosystem), U6 (cat.number 4427975 Applied Biosystem), and H19 (Hs00262142_g1 Life Technologies). Changes in the target miRNA content relative to housekeeping U6 were determined with the ΔΔct Method.

We followed previously published protocol for the extraction of RNA from FFPE tissues (11 (link)). Based on our previous experience, we used 4 × 10 μm-thick sections to extract total RNA from FFPE samples. For rapid purification, PureLink FFPE Total RNA Isolation Kit was used (Invitrogen, Thermo Fischer Scientific, Foster City, CA) according to the manufacturer's recommendations. The RNA data quality was assessed by 260/280 absorption signal ratio and the RIN number.

Total DNA was prepared by incubating overnight at 37 °C three internal FFPE sections (S3–S5) from each VSCC/MTS tissue in 250 μl of digestion buffer (10 mM Tris/HCl at pH 7.4, 0.5 mg/ml proteinase K, and 0.4% Tween 20). Then, to inactivate the proteinase K and to separate paraffin from the aqueous phase, samples were incubated at 95 °C for 10 min, centrifuged, and chilled on ice [31 (link)]. The aqueous phase was transferred to a new tube.
Total RNA was prepared from tissue sections S6–S8 using the PureLink FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA). DNase I (RNase-Free DNase Set, Qiagen, Hilden, Germany) treatment was carried out on the RNA purifying columns during sample processing as previously described [32 (link)]. Extracted RNA was eluted in 50 μl of RNase-free water and stored at − 80 °C until use.

Total RNA derived from FFPE tissues was extracted using the PureLink™ FFPE Total RNA Isolation Kit (Invitrogen) following the manufacturer's instructions and reverse-transcribed using PrimeScript RT reagent kit (Takara). The quality of the total RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington DE, USA). Quantitative PCR (qPCR) was performed using SYBR Select (Applied Biosystems) on an ABI Prism 7500 apparatus (Applied Biosystems). mRNA expression was normalized for 18S rRNA levels. Relative mRNA expression was calculated using the comparative Ct method (2−ΔΔCt). Primers used are listed in Table 3.

Ethical clearance was obtained from the study base, Mariano Marcos Memorial Hospital and Medical Center (MMMH-MC) in Ilocos Norte, Philippines. All participants gave their written informed consent. Formalin fixed paraffin embedded (FFPE) or fresh frozen biopsies from patients with histologically confirmed primary tumors of the head and neck seen at MMMH-MC between January 2003 to September 2013 were utilized in this study. Tissue sectioning, assessment of tumor content, and molecular analyses were performed at the German Cancer Research Center in Heidelberg, Germany.
FFPE and fresh frozen tissue sectioning was performed as described [12 (link), 13 (link)]. Genomic DNA from the fresh frozen sections was extracted by MagNA Pure 96 DNA and viral NA Large Volume Kit (Roche, Penzberg, Germany) following the manufacturer’s recommendations. DNA extraction from FFPE sections was done as described [13 (link), 14 (link)]. Total RNA was isolated from the fresh frozen and FFPE sections using the RNeasy Minikit (Qiagen, Hilden, Germany) and Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA), respectively. DNAse I digestion was performed prior to the last washing step to ensure exclusive amplification of RNA [14 (link)].