Rna solv

Aag2 or U4.4 cells were plated in 48-well plates and transfected with 100 ng of dsRNA per well using 0.4 μL of X-tremeGENE HD (Roche). The medium was refreshed after 3 h and cells were incubated for 48 h. For infections, cells were transfected again with dsRNA as described above, incubated for 3 h, and then infected with DENV at an MOI of 0.1. Cells were harvested at the indicated time points for total RNA isolation using RNA-Solv (Omega Bio-tek). A similar setup was used for the experiments of Fig 4G, 4I and 4L, except that cells were cultured in 24-well plates, treated twice with 150 ng of dsRNA, infected at an MOI of 0.01, after which cells or culture supernatant was harvested at 72 h post-infection, as indicated. For replicon assays, Aag2 and U4.4 cells were transfected with 100 ng dsRNA and 250 ng RepDV2Rluc RNA per well using the TransIT-mRNA transfection kit (Mirus). As a positive control, cells transfected with RepDV2Rluc RNA in the absence of dsRNA were treated with 50 μM cycloheximide (CHX) at 1 h post-transfection. Proteins were harvested in passive lysis buffer (Promega) at indicated times and Renilla luciferase activity was measured using the Renilla-Glo Luciferase Assay system (Promega).

Total RNA was obtained from NSC (3 Ctrl NSC and 3 SZP NSC) by phenol-chloroform extraction using RNAsolv (Omega Bio-Tek, Norcross, GA, USA). cDNA was then synthesized using 1 μg of RNA and a M-MLV reverse transcription kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Relative expression was assessed by qPCR (Agilent Technologies Thermocycler, Santa Clara, CA, USA) using specifically designed primers as indicated in Supplementary Table 3. Data was analyzed by calculating the expression fold change via 2^-ΔΔCt, and gene expression was normalized to that of three reference genes (GAPDH, B2M, and 18S).

Total RNA extraction was performed on DIV 21 hippocampal cultures using RNeasy Mini kit (Qiagen, Venlo, The Netherlands) and on Sprague-Dawley rat (13 weeks) hippocampus tissue using RNAsolv (Omega Bio-tek, Norcross, GA) according to manufacturer’s instructions. Genomic DNA was removed by digestion with Amplification Grade DNase I (Sigma-Aldrich). First-strand cDNA was synthesized by reverse transcription of 1 μg of total RNA using Superscript-II and random primers (Invitrogen) according to standard protocols. Reverse transcriptase was absent in some samples as negative control.

The eyelids of three female and three male human cadavers (aged 58–89 years), as well as three female and three male mice were dissected under a microscope until only the meibomian gland tissue was left. The tissues were then sliced into small pieces and placed into Lysis tubes containing 400 µL of RNA Solv (SKU: R6830-02, Omega Bio-Tek, Norcross, Georgia, USA), followed by submerging the tubes in liquid nitrogen. Homogenization was carried out in a Speedmill Plus (Analytik Jena AG, Jena, Germany). Centrifugation was used to separate the insoluble material (12,000× g, 5 min, 4 °C). Total RNA was achieved using GenElute Kit (Sigma Aldrich, Merck) according to the manufacturer’s protocol. Isopropanol and repeated ethanol precipitation was used to purify the crude RNA and an RNAse-free DNAse I (37 °C, 30 min, ThermoFisher Scientific, Waltham, MA, USA) was used to digest any contaminating genomic DNA. The DNAse reaction was stopped at 65 °C for 10 min. The generated total RNA was used to generate sample cDNA with the RevertAidTM Reverse Transkriptase-Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For each reaction, 4 µg of RNA and 2 µL of 10 pmol Oligo (dT) primers were used. The resulting cDNA was stored at −20 °C.

Total RNA was isolated using RNA-Solv (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions and treated with DNAse I. Then, the reverse transcription was performed with random primers and RevertAidTM MMuLV Reverse Transcriptase in the presence of RNAseOUT (Fermentas Glen Burnie, MD, USA). PCR reactions were performed using a 7,500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and Brilliant SybrGreen I (Stratagene). Results were analyzed with the 7,500 System Software.

RNA isolation was performed with RNAsolv (Omega Bio-tek, Norcross, USA) or SVtotal RNA Isolation System (PROMEGA, Madison, USA) according to the manufacturer’s protocols. SYBRGreen-based RT-qPCR was performed with StepOnePlus Real-Time PCR system/QuantStudio™ 3 System (Applied Biosystems, Waltham, USA) (Primers: Table S4). Expression was normalized to β-Actin.

Fertilized chicken eggs were incubated at 37.5°C with constant humidity. On the second day of incubation (E2), 2 mL of albumin was removed from the egg. On day four (E4), a rounded window was made in the shell in order to have access to the chick chorioallantoic membrane (CAM), and sealed with adhesive tape. On day ten of incubation (E10), ten million SK-N-SH shNEO1, shNTN4, or shSCR cells were drop-plated on the developing CAM. On day 17 of incubation (E17), the primary tumor of the CAM was weighed and the embryo was dissected. Embryonic lungs were incubated with RNASolv (OmegaBiotek) and kept at -20°C. We extracted genomic DNA following the manufacturer's instructions. Genomic DNA expression levels were analyzed via qPCR analysis using human Alu sequences (FW: 5′ACG CCT GTA ATC CCA GCA CTT3′; RV: 5′TCG CCC AGG CTG GAG TGC A3′) and genomic chicken GAPDH (FW: 5′GAG GAA AGG TCG CCT GG3′; RV: 5′GGT GAG GAC AAG CAG TGA3′) primers. The analysis was made using fold change with respect to chGAPDH and normalized according to lung of control cells (shSCR). Five eggs were used for each condition.