Favorprep tissue total rna mini kit

Total cell RNA was isolated using FavorPrep Tissue Total RNA Mini Kit (Favorgen) according to the manufacturer’s instructions. Eluted RNA was quantified using a NanoDrop ND100 spectrophotometer and stored at −80°C. cDNA was reverse transcribed from total RNA using MMLV reverse transcriptase (Promega) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific) on a Rotor-Gene 6000 (Corbett Research). In brief, the reaction mixture (20 μl total volume) contained 500 ng cDNA, 0.2 μM forward and reverse primers, and 5.5 μl SYBR Green PCR Master Mix. Thermal cycling conditions were 95°C for 10 s followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Experiments were performed in triplicate.

Total RNA was extracted from the viable epidermis of FTMs or epidermis and partially attached dermis of NHS using the Favorprep tissue total RNA mini kit (Favorgen, Ping-Tung, Taiwan). Methods provided by the manufacturer were followed, with a single exception of an additional 15 min DNA digestion step after loading the RNA, by means of a RNAse-free DNAse set (Qiagen, Hilden, Germany). After elution, the RNA concentration was determined using a Nanodrop™ UV-VIS spectrophotometer (Thermofisher, Waltham, MA, USA). Synthesis of complementary DNA and performance of quantitative real-time polymerase chain reactions occurred consistently with methods as described before [57 (link)]. Details on primer sequences are provided as Table S2.

Isolation of RNA from 2D and 3D cell cultures was performed according to instructions provided by the manufacturer using a FavorPrep Tissue Total RNA Mini Kit (Favorgen, Ping-Tung, Taiwan). A single exception was the incorporation of a DNA digestion step, which occured during 15 minutes after the initial washing steps using an RNAse-free DNAse solution (Qiagen, Hilden, Germany). Subsequently, the isolated RNA was eluted and the concentration of the nucleic acid content was determined using a NanoDrop™ UV-Vis Spectrophotometer (ThermoFisher).

Total RNA was extracted with FavorPrep^™ Tissue Total RNA Mini Kit (Cat No: FATRK 001; Favorgen Biotech Corp, PingTung Agricultural Biotechnology Park). First, 40 mg of tumor samples was homogenized on ice and transferred into the RNA extraction solution (Trizol) according to the manufacturer's protocol. The remaining cellular debris were removed by centrifuging (12,000 g for 10 min). The quality and purity of the extracted RNA were determined by electrophoresis on 2% agarose gel and determining the E = A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios by a Nano‐Drop spectrophotometer (Bio‐TeK). The cDNA was synthesized using 1,000 ng of the extracted RNA (BioFact™ cDNA synthesis kit, Cat. No. BR123‐R10k; BioFact^™ RT Series). The reaction mixture was prepared by admixing 1 µl of Random Hexamer primer, 10 µl of 5× reverse transcription (RT) buffer, 1 µl of oligo‐d (T), and 1 µg of total RNA and reached to a final volume of 20 µl by adding RNase‐free water. The RT reaction was carried out at 95°C (2 min), and then 60°C (30 s), and for the enzymatic reaction; the RT mixture was incubated at 74°C (4 min). The cDNA template was stored at −20°C until use (Akbari Bazm, Khazaei, & Khazaei, 2020).