Rna dna protein purification plus kit

Following treatment with atezolizumab, cells were collected from treated and non-treated wells to isolate RNA using an RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp, Ontario, Canada) as per the manufacturer’s instructions. RNA from each sample was then reverse transcribed into cDNA using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). PCR reactions were performed on QuantStudio 7 Flex qPCR (Applied Biosystems, Foster City, CA, USA) using Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). All data were normalized to β-actin. Non-specific amplifications were checked by the use of melting curve and agarose gel electrophoresis. The relative changes in target gene expression were analyzed by using the 2-ΔΔCT method. The primers were designed using Primer3 software. The sequences of primers used are as follows;

Human PD-L1 promoter forward, 5′-TGGCATTTGCTGAACGCATTT-3′.

Human PD-L1 promoter reverse, 5′-TGCAGCCAGGTCTAATTGTTTT-3′.

RNA from MSC-EP was extracted by TRIzol™ (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
Only for RNA analysis, Exoquick (System Biosciences, LLC, Palo Alto, CA, USA) was used to precipitate EVs obtained from MSC-EP (EV-EP), MSC transfected with scrambled siRNA (EV-SCR) or with the selected mimics (EV-miR127, EV-miR10a, EV-miR486, EV-miR29a). RNA was extracted with RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp, Thorold, ON, Canada), following the manufacturer’s protocol.
RNA concentration was spectroscopically determined by NanoDrop2000 (Thermo Fisher Scientific). cDNA was synthetized and RT-PCR was performed by using miRCURY™ LNA™ Universal RT microRNA PCR (Exiqon-Qiagen, Vedbaek, Denmark). Specific primers set for hsa-miR-127-3p, hsa-miR-10a-5p, hsa-miR-29a-3p, hsa-miR-486-5 were used. U6 spike-in was used for housekeeping (Exiqon-Qiagen). Data were normalized with respect to MSC transfected with the SCR and were represented as relative quantification (RQ) ± SEM.

Genomic DNA was isolated using the RNA/DNA/Protein purification plus kit (Norgen Biotek Corp, Ontario, Canada) as per the manufacturer’s instructions. The concentrations of DNA were measured using NanoDrop 2000 (Thermo Fisher Scientific, DE, USA). We used AmpliTaq™ DNA polymerase kit (Applied Biosystem, Thermo Scientific, USA) for this study. PCR was performed in a 25-μL volume, comprised 100 ng of DNA template, 2.5 μl PCR Buffer (10X), 0.5 μl dNTPs (10 mM), 0.5 μl primer mix (20 μM) and 0.125 μl AmpliTaq Gold DNA polymerase (5U/ μl). Thermal cycling was performed using the following conditions: 1 cycle 95 °C for 10 min; 35 PCR cycles 95 °C for 15 s, 55 °C for 30 s, 72 °C for 1 min; followed by final extension at 72 °C for 10 min. The PCR products were electrophoresed using a 1.5% agarose-gel/TBE buffer system using Bio-Rad. The Gel images were acquired with a CCD-gel imaging system (Bio-Rad, CA, USA).

The animals were sacrificed immediately following the last 2-h experimental session. The dissected PFC was rapidly placed on dry ice and frozen at −80 °C for further analyses.
Isolation of RNA was performed using the RNA/DNA/PROTEIN Purification Plus Kit (Norgen Biotek, Canada) following the manufacturer’s protocol. Briefly, the frozen brain structures (10–15 mg) were homogenized using the Bioprep-24 Homogenizer (Aosheng, China) (30 s at 3000 rpm, then 2 × 30 s at 2500 rpm) in the presence of ceramic beads (ø = 2.8 mm) and 350 μl of lysis buffer. RNA samples were eluted in nuclease-free water preheated to 60 °C, followed by removal of traces of DNA by treatment with DNase I (Qiagen, USA) using the RNA Clean-Up kit (Syngen, Poland) according to the manufacturer’s recommendation, after which the purified RNA was eluted in nuclease-free water and stored at −80 °C until further analysis.
The quantity and quality of the extracted RNA were determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis. Additionally, RNA integrity was evaluated using chip-based capillary electrophoresis with an RNA 6000 Nano Chip Kit and an Agilent Bioanalyzer (Agilent Technologies, USA).

The PBMC samples collected from 26 CBD and 25 patients were subjected to total mRNA extraction using an RNA/DNA/Protein Purification Plus Kit (Norgen Biotek, Thorold, Ontario, Canada). The RNA concentration was measured using Nanodrop (Nanodrop Technologies, Thermo Scientific, Inc., Wilmington, DE, USA). Further, 1.5 μg RNA was treated with 0.6 U DNAase I (Roche, Basel, Switzerland) for 30 min at 37 °C, with 8 mM EDTA for 10 min at 75 °C, and was then converted to cDNA using Superscript IV reverse transcriptase (Invitrogen, Stockholm, Sweden) in a 20 μL reaction. A reverse transcriptase negative reaction was also carried out in order to confirm the absence of genomic DNA contamination. The gene-specific primer pairs are listed in Table S1. The PCR amplification was performed using the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Gothenburg, Sweden) with an initial denaturation step of 5 min at 95 °C, followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min.

Tumor tissues (TT) and adjacent normal tissues (NT) taken away from tumor margins, were cut from freshly resected tissues by pathologist. RNA was isolated using the RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp, Thorold, Canada) as per the manufacturer’s instructions from TT and adjacent NT. Briefly, frozen tissues were transferred into a mortar containing adequate amount of liquid nitrogen and were grinded thoroughly using a pestle followed by resuspending the tissue in lysis buffer in an Eppendorf tube followed by RNA extraction protocol.

DNA and RNA were isolated using RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp, Ontario, Canada) as per manufacturer’s instructions from eight TT and their corresponding NT. Briefly, frozen tissues were transferred into a mortar containing adequate amount of liquid nitrogen and grind the tissue thoroughly using a pestle followed by resuspending with lysis buffer and collection in an Eppendorf tube. The tubes were incubated at 55 °C for 10 min followed by DNA extraction using DNA extraction column. The flow-through from DNA extraction was used for RNA and protein extractions. After DNA extraction, the RNA was extracted using RNA extraction column and the flow-through was used for protein extraction. The DNA and RNA concentrations were measured using NanoDrop 2000c (Thermo scientific, Massachusetts, USA) and stored at − 80 °C.