Total rna kit

Colonic RNA isolation was performed using the total RNA Kit (OMEGA Bio-Tek) according to the instructions. About 20 mg tissue mixed with Lysis Buffer was homogenized to extract total RNA. The purity and concentration of the extracted total RNA were tested by the NanoDropND2000 spectrophotometer (Thermo Fisher Scientific, United States). The cDNA was synthesized by the reverse transcription kit (Takara, China) at 42°C for 60 min with the oligo dT-adaptor primer according to the instruction. Real time PCR was performed with one-step qRT-PCR Kit (TOYOBO) in CFX Connect System (Bio-Rad, California, United States) with 20-µl volume, including 2 μl of cDNA, 0.8 μl of forward primer, 0.8 μl of reverse primer, 10 μl of 2 × SYBR Premix Ex Taq Ⅱ, 0.4 μl of ROX Reference Dye (50ⅹ) and 6 μl of nuclease-free water. Primer sequences were listed in Table 2. Real time PCR conditions were as follows: 5 min denaturation at 98 °C, followed by 33 cycles at 95°C at 30 s, 60°C for 30 s, and then 72 °C for 1 min. Data were analyzed using the CFX Manager software (Bio-Rad, California, United States). The data were normalized against GAPDH and expressed as fold change over mock (2^−ΔΔCt). Each sample was treated in triplicate to ensure statistical analysis significance.

Total RNA was extracted from the Vero, Marc-145, ST, BHK-21, and IPEC-J2 cells using a Total RNA Kit (Omega Bio-tek, USA) and reverse transcribed into cDNA using HiScript qRT SuperMix (Vazyme, China), following the manufacturer’s instructions. The following primers were used: PEDV N (Forward-5′-TTCTTGTTTCACAGGTGGATG-3′, Reverse-5′-GCTGCTGCGTGGTTTCA-3′); EMCV VP1 (Forward-5′-CCCCACCTCTGCTAAGATACTAAC-3′, Reverse-5′-TGGGACTGGACCTATCATAGAAG); monkey GAPDH (Forward-5′-CCTTCCGTGTCCCTACTGCCAAC-3′, Reverse-5′-GACGCCTGCTTCACCACCTTCT-3′); and porcine GAPDH (Forward-5′-TGGTGAAGGTCGGAGTGAAC-3′, Reverse-5′-AGTGGAGGTCAATGAAGGGG-3′). The detection of mRNA levels of TGEV, PRRSV, and SVA was performed as described previously [13 (link), 19 (link), 20 (link)]. Quantitative RT-PCR was performed using AceQ^® qPCR SYBR^® Green Master Mix (Vazyme, China). Each reaction was performed in triplicate and results are expressed as mean ± standard deviation (SD).

Cells stimulated with R848 or IFNs were lysed using TRK lysis buffer (Omega Bio-Tek). For in vivo infections, RNA lysates were prepared from tissue after homogenizing whole lungs. RNA was extracted using a Total RNA Kit (Omega Bio-Tek). Real-time PCR was performed after synthesizing cDNA using a High capacity cDNA Reverse Transcription kit (Applied Biosystems). The expression of Irf7 (Mm00516791_g1), Il28b (ifnl3) (Mm00663660_g1), Ifitm3 (Mm00847057_s1), Isg15 (Mm01705338_s1), Oasl2 (Mm00496187-m1), Il12b (Mm00434174_m1), Il6 (Mm00446190_m1), Tnfa (Mm00443258_m1), Ifna (Mm03030145-gH), Ccl5 (Mm01302427-m1) and Actinb (4352341E-1112017) were determined by RT-PCR (Applied Biosystems). RT-PCR reactions were performed in microAmp Fast plates (Applied Biosystems) using SensiFAST Probe Hi-ROX kit (Bioline) and a StepOnePlus RT-PCR machine (Applied Biosystems). Relative gene expression levels were calculated by normalizing the Ct levels of the target gene to both endogenous actin levels and an unstimulated WT control using the ΔΔCt method.

The mouse retinas were collected 5 days after NMDA intervention. Three individual retinas were treated as one sample, and each group contained 3 samples. RNA was isolated by total RNA kit (R6834-01, Omega Bio-Tek),. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). After RNA was converted to cDNA, the samples were sequenced by the Illumina NovaSeq 6000 at Novogene (Beijing, China). Genes with a fold-change ≥1.5 identified by edgeR and a false discovery rate <0.05 were considered differentially expressed (BMKCloud, http://www.biocloud.net/). Gene functional annotations were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) and Gene Ontology (GO, http://www.geneontology.org/) databases.

RNA was isolated from cell lines or lungs using an E.Z.N.A. Total RNA kit (Omega Bio-Tek, Norcross, GA) and converted to cDNA by high capacity cDNA reverse transcription kit (Life Technologies). Relative expression levels of c-fos, IFNα1, IFNβ1, IFNλ3, Ptges2, Reg3g, and TIMP1 were measured using probes from Applied Biosystems (Grand Island, NY) and a SensiFAST Probe Hi-ROX kit (Bioline, Taunton, MA). Samples were run on a StepOnePlus qPCR machine (Applied Biosystems), and results computed relative to WT uninfected and actin control using the ΔΔC_T method.

After treatment of 4T1 cells with various PTX formulations, the total RNA was isolated using total RNA Kit (R6934, Omega Bio-tek Inc., Norcross, GA, United States) and cDNA was synthesized from 5 μg RNA in cDNA Synthesis Kit (K1622, Fermentas International Inc., Canada) according to the manufacturer’s instructions. Expression levels of uPA, Bcl-2, twist, Survivin, CD44, MMP-2, MMP-9 and actin transcript were quantified by qRT-PCR. Each PCR was performed in triplicate in a final volume of 20 μL solution. The following forward (F) and reverse (R) primers were used in this study: uPA: 5′-GCGCCTTGGTGGTGAAAAAC-3′ (F) and 5′-GACACGCATACACCTCCGTT-3′ (R); Bcl-2: 5′-GCTACCGTCGTGACTTCGC-3′ (F) and 5′-CCCCACCGAACTCAAAGAAGG-3′ (R); Twist: 5′-GGACAAGCTGAGCAAGATTCA-3′ (F) and 5′-CGGAGAAGGCGTAGCTGAG-3′ (R); Survivin: 5′-GAGGCTGGCTTCATCCACTG-3′ (F), 5′-ATGCTCCTCTATCGGGTTGTC-3′ (R); CD44: 5′-TCGATTTGAATGTAACCTGCCG-3′ (F) and 5′-CAGTCCGGGAGATACTGTAGC-3′ (R); MMP-2: 5′-ACCTGAACACTTTCTATGGCTG-3′ (F) and 5′-CTTCCGCATGGTCTCGATG-3′ (R); MMP-9: 5′-GGACCCGAAGCGGACATTG-3′ (F) and 5′-CGTCGTCGAAATGGGCATCT-3′ (R); actin: 5′-GGCTGTATTCCCCTCCATCG-3′ (F) and 5′-CCAGTTGGTAACAATGCCATGT-3′ (R). Assays were performed in triplicate with the ABI 7500 instrument (Waltham, MA, United States). All data were normalized by actin. Gene expression data was analyzed by the 2^-ΔΔCT method.