From each plant, we sampled two mature leaves, which were flash frozen in liquid nitrogen and stored at −80°C until DNA and RNA extraction. DNA and RNA extractions were done using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the NucleoSpin RNA Plus Mini kit (Macherey-Nagel, Düren, Germany), respectively, following the protocols provided by the manufacturers. Contaminating genomic DNA was removed from the RNA samples by treatment with the TURBO DNA-Free Kit (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was then synthesized using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) based on random hexamer priming according to the manufacturer’s instructions.
Nucleospin rna plus mini kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoSpin RNA Plus Mini kit is a laboratory product designed for the isolation and purification of total RNA from various biological samples. It employs a silica-membrane technology to efficiently capture and purify RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy plant mini kit, by Qiagen (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Transcriptor high fidelity cdna synthesis kit, by Roche (1 mentions)
Infinite f200 pro plate reader, by Tecan (1 mentions)
Steponeplus fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rt2 qpcr primer assay, by Qiagen (1 mentions)
Celltiter glo, by Promega (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Qpcrbio sygreen mix hi rox kit, by PCR Biosystems (1 mentions)
Qpcrbio probe mix hi rox kit, by PCR Biosystems (1 mentions)
Dnase, by Macherey-Nagel (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Fastprep 24 homogenizer, by MP Biomedicals (1 mentions)
Lightcycler 480 probes master mix, by Roche (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Random primer, by Roche (1 mentions)
9 protocols using nucleospin rna plus mini kit
1
Nucleic Acid Extraction and cDNA Synthesis
2
Quantifying SARS-CoV-2 Viral RNA Levels
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Cellular viability was measured using Cell Titer Glo (Promega), according to the manufacturer’s instructions. Luminescence was measured on a TECAN infinite F200PRO plate reader. Viral RNA from supernatants were isolated from 100 µL supernatant using the QIAamp Viral RNA Mini Kit (Qiagen) and viral RNA from cell lysate was extracted using the Nucleospin RNA plus mini kit (Macherey-Nagel), according to the manufacturer’s instructions. Ten microliters of RNA were used to synthesize cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. GAPDH transcripts were detected by RT2 qPCR Primer Assay (Qiagen, Cat# 330001 PPQ00249A) and the qPCRBIO SyGreen Mix Hi-ROX kit (PCRBIOSYSTEMS); viral transcripts were detected using the qPCRBIO Probe Mix Hi-ROX kit (PCRBIOSYSTEMS) and the indicated primers and probes (Table 1 ). For strand specific qPCR the random primers of High Capacity cDNA Reverse Transcription Kit were replaced with either the forward or reverse SARS-CoV-2 primer. The qPCR was run on a StepOnePlus fast real-time PCR system (Applied Biosystems).
3
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated using the NucleoSpin RNA Plus Mini Kit (Macherey-Nagel, Düren, Germany), followed by treatment with DNase (Macherey-Nagel, Düren, Germany) to remove genomic DNA. Total RNA (1 µg of the total RNA) was reverse-transcribed using the High-Capacity Reverse Transcription kit (Applied Biosystems, Inc., Foster City, CA, USA), and qRT-PCR was conducted on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SsoAdvanced Universal SYBR green supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The sequences of the primers used are shown in Table S5 .
4
RNA Extraction from Tissue Homogenate
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Tissue was disrupted in Lysing matrix D tubes containing 600 μL LBP (NucleoSpin kit) with a FastPrep 24 homogenizer (MP Biomedicals GmbH, Eschwege, Germany; 3 × 40 s). Debris was spun down, the supernatant was recovered and cleared using Qia-shredder columns (Qiagen GmbH, Hilden, Germany). Total RNA was extracted from this eluate using the NucleoSpin RNA Plus mini kit from Macherey–Nagel (Düren, Germany) according to the instructions of the vendor.
5
Intestine Gene Expression Analysis
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The intestine from adult Neurog3ΔAdInt and control males was collected 18 weeks (males under CD) and 5 weeks (males under HFD) after tamoxifen administration. Total RNA from duodenum, jejunum, ileum, proximal colon, or distal colon was extracted using NucleoSpin RNA Plus, Mini Kit (Macherey-Nagel) according to the manufacturer’s instructions. Reverse transcription was performed using Transcriptor Reverse Transcriptase (Roche) and Random primers (Roche). Quantitative polymerase chain reactions were performed in a Light Cycler 480 (Roche) with Light Cycler 480 Probes Master Mix (Roche) using mouse-specific TaqMan primers and probes (ThermoFisher) recognizing Neurog3 (Mm00437606_s1), Cck (Mm00446170_m1), ChgA (Mm00514341_m1), Defa1 (Mm02524428_g1), Gcg (Mm00801712_m1), Ghrl (Mm00445450_m1), Gip (Mm00433601_m1), mKi67 (Mm01278617_m1), Lgr5 (Mm00458299_m1), Lyz1 (Mm00657323_m1), Muc2 (Mm00458299_m1), Nts (Mm00481140_m1), Olfm4 (Mm01320260_m1), Pcna (Mm00448100_g1), Pyy (Mm00520715_m1), Sct (Mm00441235_g1), Sst (Mm00436671_m1), Tph1 (Mm00493794_m1). Gene expression levels were normalized to Actb (4352933E) or Rplp0 (Mm01974474_gH). Relative changes in gene expression were determined by the 2-ΔΔCt method.
6
Gene Expression Analysis of Cytokines
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Total RNA was extracted with the NucleoSpin RNA Plus, Mini kit (Macherey-Nagel, Düren, Germany, # 740984.50) according to the manufacturer’s protocol. RNA concentrations were evaluated using a spectrophotometer, and the purity was assessed based on the ratio of absorbance at 260 nm and 280 nm (A260–A280). Subsequently, RNA was reversely transcribed using the qScript Ultra SuperMix (Quantabio, Beverly, MA, United States, #95217-100) and the resulting cDNA was used for real-time polymerase chain reaction (PCR) with the PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, United States, # 95071). The gene expression of IL-6, Cyr61/CCN1, and CTGF/CCN2 was quantified by real-time PCR on the PCR MyiQ™ 2 Cycler (Biorad, Kabelsketal, Germany), using the IQ 5 software (version: 2.1.97.1001) and the following conditions: 95°C for 15 s, followed by 45 cycles at 95°C for 5 s, 60°C for 15 s, and extension at 68°C for 10 s. The primers (Eurofins Genomics, Ebersberg, Germany) are listed in Table 1 . Cycle threshold (Ct) values of target genes were normalized to the housekeeping gene β-Actin.
7
Quantitative Gene Expression Analysis
8
RNA Extraction and qPCR Analysis
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Six replicate RNA preparations for each genotype or condition were prepared using the NucleoSpin RNA Plus Mini kit (Macherey-Nagel, #740984.50) kit; each sample contained 3-5 whole late feeding third-instar larvae or 5 larval brains. Animals or tissues were placed in 2-mL Eppendorf tubes containing lysis buffer + 1% betamercaptoethanol, and samples were lysed using a bead mill (Qiagen). RNA was purified according to the kit instructions, and cDNA was prepared using the High-Capacity cDNA Synthesis Kit (ThermoFisher, #4368814). QPCR reactions were prepared using RealQ Plus 2x Master Mix Green without ROX (Ampliqon, #A323402) and the gene-specific primers given in the STAR methods, and runs were performed using a QuantStudio 5 machine (Applied Biosystems).
9
Total RNA Isolation and cDNA Synthesis
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For total RNA isolation, the cells were washed with icecold PBS 24-h post-LPS treatment. RNA was extracted with spin column followed by NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column (Macherey-Nagel) according to the manufacturer's instructions. RNA (0.5 µg per sample) was reverse transcribed to cDNA using a first-strand cDNA synthesis kit (High Capacity, ThermoFisher Scientific) following the manufacturer's protocol. For real-time qPCR studies, the cDNA samples were diluted 50 times with sterile miliQ water.
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