Cells were lysed and total RNA was extracted with Nucleospin RNA II Kit (Macherey-Nagel). Muscle tissues were first lysed with Trisure reagent (Bioline) and RNA was further extracted with the Nucleospin RNA II Kit (Macherey-Nagel). cDNA synthesis was performed with the iScript cDNA synthesis kit (Bio-Rad) using 1 μg RNA and random hexamer primers. qRT-PCR was performed with the iQ SYBR green supermix (Bio-Rad). Relative gene expression levels were calculated using the formula 2−ΔΔCt. Primers are listed in Supplementary Table 1 .
Nucleospin rna 2 kit
Manufactured by Macherey-Nagel
Sourced in Germany, France, United States, Switzerland, Japan, Sweden
The NucleoSpin RNA II kit is a laboratory tool designed for the isolation and purification of total RNA from a variety of biological samples. It utilizes a silica-membrane technology to efficiently capture and purify RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (51 mentions)
Iscript cdna synthesis kit, by Bio-Rad (46 mentions)
Trizol, by Thermo Fisher Scientific (33 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (29 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (28 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (26 mentions)
Trizol reagent, by Thermo Fisher Scientific (24 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (24 mentions)
Nanodrop, by Thermo Fisher Scientific (23 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (23 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (22 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (22 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (20 mentions)
Iq sybr green supermix, by Bio-Rad (19 mentions)
Lightcycler 480, by Roche (18 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (17 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (16 mentions)
Tri reagent, by Merck Group (14 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (14 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (14 mentions)
814 protocols using nucleospin rna 2 kit
1
RNA Extraction and qRT-PCR from Muscle Tissue
2
Colonic Gene Expression Analysis
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Colonic tissue samples were homogenized with ceramic beads using Precellys Lysing Equipment (Bertin Technologies, Montigny le Bretonneux, France, ref. P000911-LYSK0-A). Total RNA was extracted from colonic samples with NucleoSpin RNAII kits (Macherey-Nagel, Hoerdt, France, ref. 740955). The complementary DNA was prepared with High-Capacity Complementary DNA Archive kits (Thermo Fisher Scientific, Villebon-sur-Yvette, France, ref. 4368813). Transcripts levels of genes involved in inflammation and pain transduction were quantified in the StepOne real-time polymerase chain reaction (PCR) system using a SYBR Green PCR master mix (Thermo Fisher Scientific, ref. 4385612). Relative messenger RNA (mRNA) levels were determined using the ΔΔCt method and the values were normalized to the expression of PolR2a for mice and Gapdh for rats.52 Primer sequences are available upon request.
3
RNA Isolation and qPCR Analysis
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Total RNA was isolated using Nucleospin RNA II kits (Macherey-Nagel GmbH, Germany). Quantitative PCR (qPCR) reactions were performed using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) with KAPA SYBR® FAST qPCR Master mix. Actin was used as an internal control. The primer sequences used for qPCR are provided in Supplementary Table 1.
4
Isolation and Cloning of Human Antibody Variable Regions
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RNA and gDNA were prepared either using the “NucleoSpin Tissue” and “NucleoSpin RNA II” kits (Macherey–Nagel, Düren, Germany) or the “RNeasy Plus Mini” kit (Qiagen, Hilden, Germany). Messenger RNA (mRNA) from LCLs was reverse transcribed using the “SuperScript III” kit (Life Technologies). Human heavy chain and light chain variable regions (Vh and Vl regions) from LCLs were recovered as described before, with minor modifications [23 (link)]. Our approach differed in that mRNA from B cell clusters instead of from single cells was used. Moreover, (1) a re-amplification step with the primer set for the first PCRs was included and (2) these sequences were cloned into a TopoVector (pCR®II-TOPO®, Life Technologies) in order to be able to remove potential endogenous cleavage sites. All kits were used according to the manufacturers’ instructions.
5
Quantitative RT-PCR Analysis of Inflammatory Cytokines
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Total RNA was purified from the bilateral frontal cerebral cortex and right inferior lobe of lung using Nucleospin® RNA II Kits (Macherey-Nagel, Duren, Germany) in accordance with the manufacturer's instructions. First-strand cDNA synthesis and qRT-PCR were performed using a One Step TB Green PrimeScript™ RT-PCR Kit II (Takara Bio, Shiga, Japan). Subsequently, qRT-PCR assays were conducted using a Thermal Cycler Dice Real-Time System II TP 900 (Takara Bio). The primers for mouse 18S rRNA and IL-6 genes were purchased from Qiagen (Valencia, CA; Catalog Numbers: 18S mouse, QT02448075; IL-6 mouse, QT00098875). The primers targeting IL-1β and TNFα were purchased from Invitrogen (CA, USA) and the primer targeting IL-1α was purchased from FASMAC (Kanagawa, Japan). The primer sequences utilized were the following: IL-1α 5′-AAGACAAGCCTGTGTTGCTGAAGG-3′ (forward) and 5′-TCCCAGAAGAAAATGAGGTCGGTC-3′ (reverse); IL-1β 5′-CGACAAAATACCTGTGGCCT-3′ (forward) and 5′-TTCTTTGGGTATTGCTTGGG-3′ (reverse); TNFα 5′-TCGTAGCAAACCACCAAGTG-3′ (forward) and 5′-CCTTGAAGAGAACCTGGGAGT-3′ (reverse). The delta CT method was used to calculate the relative fold change of targeted genes. For each target mRNA, the fold changes in expression were calculated relative to those of 18S rRNA, and the value of treated group was normalized that of each control group.
6
Hippocampal RNA Extraction and qPCR Analysis
7
Total RNA Extraction from Brain Regions
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Total RNA was extracted from PFC, NAC, and HIPP by using Nucleospin RNA II kits (Macherey-Nagel, Dublin, Ireland) according to the manufacturer's instructions. Total RNA concentrations were measured with a NanoDrop® (Thermo Scientific, Dublin, Ireland) micro-volume spectrophotometer and all RNA samples were equalized with RNase-free water to the lowest detected concentration.
8
Quantitative RT-PCR for Interferon Genes
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RNA was extracted from 0.4 to 1 million cells using the Nucleospin RNA II kits (#740955.50; Macherey-Nagel). cDNA was synthesized using random primers (#48190-011; Invitrogen) from 0.1 μg RNA using Superscript III Reverse transcriptase (#18080044; Thermo Fisher Scientific). Real-time qPCR (RT-qPCR) was carried out in 20 μl reactions using SYBR Green I Master (#4887352001; Roche). Primers used were as follows: IFNL1 (Fwd: 5′-GGTGACTTTGGTGCTAGGCT, Rev: TGAGTGACTCTTCCAAGGCG-3′), IFNB (Fwd: 5′-GTCTCCTCCAAATTGCTCTC, Rev: ACAGGAGCTTCTGACACTGA-3′), ACTB (Fwd: 5′-GGACTTCGAGCAAGAGATGG, Rev: AGCACTGTGTTGGCGTACAG-3′), B2M (Fwd: 5′-TCTCTGCTGGATGACGTGAG, Rev: TAGCTGTGCTCGCGCTACT-3′).
9
Ileum Total RNA Extraction Protocol
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Total RNA was isolated from ileum at 35 and 49 days of age (n = 57 rabbits) as previously described36 (link). Ileum samples were homogenized and grounded to a fine powder by rapid agitation for 1 min in a liquid-nitrogen cooled grinder with stainless steel beads. An aliquot of 100 mg of the fine powder was then processed for total RNA isolation and purification using Trizol (Invitrogen, France) and the Nucleospin RNA II kit (Macherey–Nagel, France) according to the manufacturer’s instructions. The method included a DNase digestion step to remove contaminating DNA. The extracted total RNA was eluted in 70 μl of RNase-free water and stored at − 80 °C. RNA quality and concentration were controlled using an AGILENT 2100 bioanalyzer (RNA solutions and RNA 6000 Nano Lab- Chip Kit, Agilent Technologies France, Massy, France).
10
Polysome Profiling of Mammalian Cells
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Polysome profiling experiments were performed according to Verrier and Jean-Jean [29 (link)] with minor modifications. Three days after electroporation, two 100-mm plates of either HCT116 or HEK293 cells at 70% cell confluence were used for polysome fractionation. Cell extracts were loaded onto 15–50% sucrose gradients and subjected to ultracentrifugation. After centrifugation, optical density (O. D.) at 254 nm was monitored by pumping the gradient through a Retriever 500 (Teledyne Isco) fraction collector. The flow rate was optimized in order to collect ~15 fractions of 0.8 mL each. Afterward, each fraction was precipitated with 1.2 mL of isopropanol and kept at −80°C for further RNA analysis. For RNA isolation, the polysomal fractions of the sucrose gradients (Figure 1 ) were subjected to centrifugation at 18,000 × g for 1 hr, then the pellets were rinsed with 1 mL of 80% ethanol, dried and resuspended in 350 μl of RAI buffer of NucleoSpin RNA II kit (Macherey-Nagel) containing 1% ß-mercaptoethanol (v/v). RNA was purified according to the manufacturer's instructions and eluted in 40 μl of RNase-free water.
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