The extraction of RNA was performed from VICs using the Total RNA Purification Plus Kit (Norgen Biotek Corp, Thorold, ON, Canada). RNA was quantified using Nanodrop and used for two-step PCR amplification with a TaqMan Reverse Transcription Reagent kit (Life Technologies, Carlsbad, CA, USA). Total RNA (1 µg) was converted into cDNA. Real-time PCR (qPCR) was performed on ABI Prism 7900 HT (Applied Biosystems, Waltham, MA, USA), according to the manufacturer’s instructions, and analysis was performed using SDS2.4 software (Life Technologies, Carlsbad, CA, USA). Ribosomal Protein L32 (RPL32) was used as a housekeeping gene.
Total rna purification plus kit
Manufactured by Norgen Biotek
Sourced in Canada
The Total RNA Purification Plus Kit is a laboratory equipment designed for the extraction and purification of total RNA from a variety of sample types. It utilizes a fast and efficient spin-column based protocol to isolate high-quality RNA suitable for various downstream applications.
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Lab products found in correlation
Sensifast cdna synthesis kit, by Meridian Bioscience (6 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (5 mentions)
Rotor gene 3000, by Qiagen (5 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Lifepro thermal cycler, by Bioer (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Taqman reverse transcription reagent kit, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Hiseq 4000 platform, by Illumina (3 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix, by Roche (3 mentions)
Transcriptor universal cdna master kit, by Roche (3 mentions)
Corbet rotor gene 6000 qpcr machine, by Qiagen (3 mentions)
Sensifasat sybr no rox kit, by Meridian Bioscience (3 mentions)
98 protocols using total rna purification plus kit
1
RNA Extraction and qPCR Analysis of VICs
2
Plasma Total RNA Extraction
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Total RNA was extracted from plasma using the Total Rna Purification Plus Kit (Norgen, Thorold, ON, Canada), according to the manufacturer’s instructions.
3
Characterization of Idiopathic Pulmonary Fibrosis Mesenchymal Stem Cells
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Tissue fragments were cultured with MSCGM medium (Lonza, Basel, Switzerland) [43 (link)]. MSCs isolated from patients affected by idiopathic pulmonary fibrosis were named IPF-MSCs, while MSCs isolated from control lung of patients with adenocarcinoma were defined C-MSCs. According to the criteria by Dominici [20 (link)], cells were characterized by testing the plastic adherence, the immunophenotype and the multipotency as previously described [21 (link),44 (link)]. Finally, the expression of genes related to stemness (OCT4, SOX2, NANOG, KFL4) was analyzed by qRT-PCR. Cell pellet was collected from 3 × 105 cells after 72 h of culture and total RNA was extracted with Total RNA Purification Plus Kit (Norgen, Biotek Corp, Schmon Parkway, Thorold, ON, Canada). cDNA synthesis was performed using 5× All-In-One RT MasterMix (Applied Biological Materials, Richmond, BC, Canada). After amplification qRT-PCR was carried out with SsoFast EvaGreen Supermix (Bio-Rad, Milano, Italy) and fluorescence and melting curves were acquired. The parameter threshold cycle (Ct) was defined as the cycle number at which the first detectable increase above the threshold in fluorescence was observed. All samples were tested in duplicate with the housekeeping genes GAPDH for data normalization. mRNA expression was calculated by the 2−ΔΔCt method [45 (link)]. The primer sequences are summarized in Table 3 .
4
Transcriptional Profile Analysis of HC and ACM C-MSC
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Total RNA was extracted from HC and ACM C-MSC using the Total RNA Purification Plus kit (Norgen Biotek Corp., Thorold, ON, Canada) and reverse transcribed by SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Then qRT-PCR was performed in triplicate using 15 ng of cDNA and the iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). All these processes were performed following manufacturers’ instructions. The primer sequences of analyzed genes are reported in Table 1 .
5
Quantifying Gene Expression in Tissue Engineered Constructs
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Total RNA was isolated from cells grown onto scaffolds or onto plastic for 7 days using the total RNA purification Plus kit (Norgen Biotek, Toronto, ON, Canada). The RNA quality and concentration of the samples were measured with the NanoDrop™ ND-1000 (Thermo Fisher Scientific). For each sample, 500 ng of total RNA was reverse-transcribed using an RT2 First Strand kit (Qiagen, Hilden, Germany) in a final reaction volume of 20 μL [39 (link)]. Real-time PCR was performed according to the user’s manual on wound healing or inflammatory response and Autoimmunity RT2 profiler PCR Array (Qiagen) or the primer reported on Table 1 with a StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Foster City, CA, USA) using RT2 SYBR Green ROX FAST Master Mix (Qiagen). Thermal cycling and fluorescence detection were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. At the end of each run, a melting curve analysis was performed using the following program: 95 °C for 1 min, 65 °C for 2 min with optics off, and 65 °C to 95 °C at 2 °C/min with optics on. Each experiment was repeated 3 times, and each measure was repeated 3 times [55 (link)].
6
Total RNA Extraction from Cells
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We isolated total RNA from 300.000 to 600.00 cells with the Total RNA Purification Plus Kit (Norgen Biotek, Thorold, ON, Canada). We measured the concentration and quality of the total RNA in each sample on a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). We froze the total RNA at −80 °C until analysis.
7
Assessing RXRA Expression in CML
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RNA was extracted from CML CD34+ cells (n = 39) and healthy donor CD34+ cells (n = 9) using total RNA purification plus kit (Norgen Biotek Corp, Canada). Expression of RXRA was assessed using nCounter, and data counts were normalized with housekeeping genes–ACTB, GAPDH & GUS using nSolver software (NanoString, Technologies, Seattle, WA).
8
Quantitative Expression Analysis of Inflammatory Genes
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RNA was isolated using the Total RNA Purification Plus Kit (Norgen Biotek, ON, CA) according to the manufacturer's instructions. For cDNA synthesis, 1 μg total RNA was transcribed with cDNA transcription reagents using the High Capacity cDNA Reverse Transcription Kit (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer's instructions. Gene expression was measured in real-time PCR performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Primer sequences (forward and reverse, resp.) were the following: human TGF-β 5′-GACACCAACTATTGCTTCAG-3′ and 5′- CAGGCTCCAAATGTAGGG-3′, TNF-α 5′-CCCAGGGACCTCTCTCTAATCA-3′ and 5′-GGTTTGCTACAACATGGGCTACA-3′, IL-1β 5′-TGATGGCTTATTACAGTGGCAATG-3′ and 5′-GTAGTGGTGGTGGGAGATTCG-3′, IL-6 5′-CAATCTGGATTCAATGAGGAGAC-3′ and 5′-TGTTCCTCACTACTCAAATCT-3′, IL-8 5′-TGGCAGCCTTCCTGATTTCT-3′ and 5′- GGGTGGAAAGGTTTGGAGTATG-3′ and CCL-3 5′-CCGGTGTCATCTTCCTAACC-3′ and 5′-TTCTGGACCCACTCCTCACT-3′, and GAPDH 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′- GAAGATGGTGATGGGATTTC-3′. The expression level of a gene in a given sample was represented as 2−ΔΔCt where ΔΔCT = [ΔCT(experimental)] − [ΔCT(medium)] and ΔCT = [CT(experimental)] − [CT(housekeeping)]. The GAPDH levels were used to normalize gene expression levels.
9
Adipocyte RNA Isolation and Sequencing
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Adipocytes were isolated from human VAT as previously described (29 (link)). Total RNA was isolated with Total RNA Purification Plus Kit (Norgen Biotek, Canada). RNA quality and quantity was assessed by Agilent 2,100 Bioanalyzer and samples stored at −80°C until use. Total RNA (2 μg) was used to prepare the library for Illumina sequencing (Illumina TruSeq Small RNA Sample Preparation). Single-end reads (>10 M reads per sample) were produced by Illumina HiSeq 2000.
10
Quantitative RT-PCR Analysis of Tβh and tdc2 in Starved Flies
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For quantitative RT-PCR, brains were dissected from 24 h starved or non-starved Canton-S male flies (n = 20) and total RNA was extracted using Total RNA Purification Plus Kit (Norgen Biotek). 1 ng-1 μg RNA was reverse transcribed to cDNA using FastGene Scriptase II cDNA synthesis kit (Nippon Genetics) using oligo dT primers in accordance with the manufacturer’s protocol. SensiFAST SYBR Hi-ROX Kit (Bioline) and primers targeting Tβh or tdc2 were used to perform quantitative real-time PCR on ABI PRISM 7000 Sequence Detection System. Each experiment was performed in technical triplicates. Rp15 was used as an internal control for normalization. Table S1 lists the sequence of primers used.
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