Mirneasy mini kit 50

Paired tumor and non-malignant adjacent mucosa samples were obtained from 50 patients who underwent surgery between the years 2011 and 2015 and in whom all information was followed and updated in 2021 (patients’ characteristics in Table 1 and Supplementary Table 1). All the patients provided signed consent for participation and their medical documentation for research. The design of the study was approved by the Ethical Committee of the Institute of Experimental Medicine, Prague, Czech Republic. RNA was isolated from tissues by miRNeasy^® Mini Kit (50) (Qiagen, Hilden, Germany).

C6/36 cells were infected with DENV-1 and ZIKV at a multiplicity of infection (MOI) of 0.25. At 3 days post infection (dpi), the culture medium was removed, and total RNA extraction was carried out using miRNeasy Mini Kit 50 (Qiagen, 217004) according to the manufacturer’s protocol. RNA concentration was determined using ND-2000 instrument (NanoDrop Technologies), and RNA integrity was determined using 2100 Bioanalyzer (Agilent). Libraries were constructed using Illumina TruSeq RNA Sample Prep kit (Illumina, San Diego) using one microgram (μg) of total RNA as starting material. The prepared libraries were sequenced on the Illumina HiSeq 2000 platform to generate 150 bp paired-end reads. Raw reads were deposited in NCBI with accession SRP221722 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP221722).

C6/36 cells were infected with DENV1 at multiplicity of infection (MOI) of 0.25. Three days after infection, RNA extraction was carried out using miRNeasy Mini Kit 50 (Qiagen, Hilden, Germany, 217004) according to the manufacturer’s protocol. Total RNA was then subjected to next-generation sequencing. The RNA-sequencing libraries were prepared using standard Illumina protocols and sequenced using the HiSeq-SE50 platform, generating paired-end reads of 50 bp in size.

To extract the total RNA, the High Pure RNA Isolation Kit (miRNeasy, Mini Kit 50, QIAGEN) was utilized following the kit protocol. The extracted RNA from the soybean leaf samples was analyzed for its quantity, purity, and integrity using NanoDrop (Boeco, Germany) by measuring the OD_260/280. The concentration of all extracted RNA samples was adjusted to the same level using RNAase-free water before the next step.

The samples collected for RNA extraction were stored in RNAlater^® Tissue Collection (Ambion, Woodlands, TX, USA) at −20 °C. RNA extraction was performed using miRNeasy^® Mini Kit 50 (Qiagen, cat. No 217004) according to the manufacturer’s protocol. The quality of RNA was confirmed using a Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA).
Complementary DNA (cDNA) synthesis from total RNA was performed with the High-capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA) for expression analysis of TRAIL; the cDNA synthesis for microRNA analysis was performed using the TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems™, USA). Both procedures were performed according to the manufacturer’s instructions.
The expression was determined by the ABI Prism 7500 Fast Sequence Detection System using TaqMan assays (Applied Biosystems). The qPCR used TRAIL (Hs00366278_m1) and hsa-miR-106b-5p (000442) as target genes. The reference genes for normalization were UBC (Hs00221499_m1) and TBP (Hs00187332_m1) for TRAIL, and for miR-106b-5p, RNU6B (Hs001093) and RNU48 (Hs001006) were employed. The relative quantification of the expression was calculated using the 2^−ΔΔCt method [28 (link)].