Rna extraction mini kit

RNA extraction was performed at different stages of differentiation (days 0, 1, 3, and 7) using a Qiagen RNA extraction minikit (Qiagen), according to the manufacturer’s protocol. RNA concentration was determined using a Nanodrop spectrometer, and 500 ng of RNA was used for cDNA synthesis using the Takara PrimeScript RT reagent kit, following manufacturer’s instruction. Real-time PCR was performed using an SYBR green assay on a 7900HT SDS system from ABI. The efficiency of each primer was verified with serial dilutions of cDNA. Relative expression levels were calculated by normalization to the geometric mean of the three housekeeping genes (Eef1a, Tubb, and Actb). The highest normalized relative quantity was arbitrarily designated as a value of 1.0. Fold changes were calculated from the quotient of means of these RNA normalized quantities and reported as ±SEM. The list of primers is displayed in Supplementary Table 2.

Gag, Pol, and Nef sequences were generated from either viral RNA or genomic DNA. DNA was extracted from whole blood, and viral RNA was extracted from plasma using an RNA extraction minikit (Qiagen UK) in accordance with the manufacturer's instructions. Reverse transcription of RNA to cDNA was undertaken using a Superscript III one-step reverse transcriptase kit (Invitrogen) as a one-step reaction combined with outer PCR according to the manufacturer's instructions and amplified by nested PCR to obtain population sequences. Sequencing was undertaken using the BigDye Ready Reaction Terminator Mix (V3) (Applied Biosystems UK) analyzed using Sequencher v4.8 (Gene Codes Corporation) and manually aligned using Se_Al software.

Total RNA extraction, cDNA synthesis, and gene expression analyses were performed. RNA extraction (RNA extraction mini kit, Qiagen, Hilden, Germany) from the colonic tissue was performed according to the manufacturer′s protocol. The primers and FAM-labeled probe sequences of TNF-α, IL-1β, IL-6, and β-actin used in this study are listed in Table 2.
Real-time PCR amplification was performed using the Maxima Probe qPCR master mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) with an ABI Prism 7500 fast instrument (Applied Biosystems, Foster city, CA, USA). Briefly, primers of target genes, probes, and cDNA samples were added into the Maxima Probe qPCR master mix. The reactions were performed by pretreatment with uracil-DNA glycosylase at 50 °C for 2 min and an initial preincubation at 95 °C for 10 min, followed by 40 amplification cycles, as follows: 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The gene expression levels of the genes of interest were normalized to β-actin, and the results were analyzed according to the ΔΔCt method. We reported the copy number change of the therapy group compared with that of the control group.

Total RNA was extracted from bone marrow cells using RNA extraction mini kit (QIAGEN). cDNA synthesis was carried out using iScript Reverse Transcription Supermix (#170-8841, Bio-Rad). Primer sequences were presented in the supplementary materials.
Quantitative RT-PCR was performed using the SsoAdvanced Universal SYBR Green Supermix (#172-5124, Bio-Rad) and run on standard cycle conditions followed the manufacturer's protocol.

Total RNA was extracted from fibroblasts, MDA-MB-231, MDA-MB-361 and MDA-MB-453 cells using the RNA extraction mini kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instruction. Two-step RT-qPCR was performed to assess the mRNA level of SOD1, SOD2, CAT, PRDX1 and GSR. First strand cDNA was synthesized using iScriptTM cDNA Synthesis Kit (BioRad, Hercules, CA, USA). q-PCR was set up using CFX96 Real-Time System (BioRad). Actin was used as an internal control. Paired primer sequences used were SOD1: 5′-GGTGGGCCAAGGATGAAGAG-3′ (forward) and 5′-CCACAAGCCAACGACTTCC-3′ (reverse); SOD2: 5′-GCTCCGGTTTTGGGGTATCTG-3′ (forward) and 5′-GCGTTGATGTGAGGTTCCAG-3′ (reverse); CAT 5′-TGGAGCTGGTAACCCAGTAGG-3′ (forward) and 5′-CCTTTGCCTTGGAGTATTTGGTA-3′ (reverse); PRDX1 5′- CCACGGAGATCATTGCTTTCA-3′ (forward) and 5′-AGGTGTATTGACCCATGCTAGAT -3′ (reverse); GSR: 5′-CACTTGCGTGAATGTTGGATG-3′ (forward) and 5′-TGGGATCACTCGTGAAGGCT-3′ (reverse); and Actin: 5′AAGGAGCCCCACGAGAAAAAT-3′ (forward); 5′- ACCGAACTTGCATTGATTCCAG-3′.

Total RNA was extracted from parental and TDR cells treated with DMSO or dabrafenib + trametinib using the RNA extraction mini kit (Qiagen, cat#74104, Hilden, Germany) according to the manufacturer’s instructions. The RNA concentration was determined using a NanoDrop™ (ThermoScientific, Waltham, MA, USA) spectrophotometer. Two-step RT-qPCR was performed to assess the mRNA levels. First strand cDNA was synthesized using iScriptTM cDNA Synthesis Kit (BioRad, cat#1708891, Hercules, CA, USA). The mRNA levels were quantified by qPCR using primers purchased from Integrated DNA Technologies. qPCR was set up using CFX96 Real-Time System (BioRad). The relative mRNA levels were calculated by the 2^−ΔΔCt method. Actin was used as internal control. Primers used for the experiment are listed in Table S2.

To produce dsRNA, the bacteria transformed with L4440-SeINT were grown in Luria-Bertani (LB) containing 100 μg/mL ampicillin (LB-AMP) at 37^°C for 16 h with 250 rpm shaking rate. The cultured broth (5 mL) was added to 500 mL of LB medium and allowed to grow to OD₆₀₀ = 0.6~0.7. Expression of T7 RNA polymerase gene was induced by an addition of IPTG to 0.1 mM and the bacteria were incubated with shaking for 4 h at 37°C. The expressed dsRNA was extracted using a RNA extraction mini kit (Qiagen Korea, Seoul, Korea). The purity of the synthesized dsRNA was confirmed by electrophoresis on 1% agarose gel. To assess insecticidal activity of the recombinant bacteria expressing dsRNA, IPTG-induced cultures were pelleted by centrifugation and resuspended in the same culture medium, and applied to cabbage leaves for feeding treatment (see below).