GFP positive HEK 293T cells, transfected with either pEGFP, pEGFP-E1, were sorted by flow cytometry (BD FACSAria™ II, USA), and DNA, RNA, and protein from the sorted cells were extracted using NucleoSpin® TriPrep (Macherey-Nagel, Germany). Extracted RNA was then precipitated using 0.3M sodium acetate and 1 volume of isopropanol. The solution was chilled at -20°C for at least 1 hour. The pellet was centrifuged for 15 minutes (15,000 g, 4°C), washed with 70% ethanol and centrifuged again for 15 minutes (15,000 g, 4°C). Precipitated RNA was then sent for microarray analysis (Macrogen, Republic of Korea).
Nucleospin triprep
Manufactured by Macherey-Nagel
Sourced in Germany
NucleoSpin® TriPrep is a kit for the simultaneous extraction of DNA, RNA, and proteins from a single sample. It employs silica-membrane technology to efficiently capture and purify the various biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (2 mentions)
Hg u133 plus 2.0 array, by Thermo Fisher Scientific (2 mentions)
Qiasymphony rna, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Dneasy isolation kit, by Qiagen (1 mentions)
Cryolys cooling system, by Bertin Technologies (1 mentions)
Precellys 24 homogenizer, by Bertin Technologies (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Forward and reverse primer, by Thermo Fisher Scientific (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green reagent, by Thermo Fisher Scientific (1 mentions)
Sds software, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
20 protocols using nucleospin triprep
1
Comprehensive Cell Analysis via FACS
2
RNA Extraction, Microarray, and Differential Gene Expression Analysis
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RNA was extracted with the Nucleospin TriPrep (Macherey-Nagel) and the QIASymphony RNA (Qiagen) kits according to the manufacturers’ instructions. Affymetrix HG U133 plus 2.0 arrays were prepared and scanned according to the manufacturer’s protocol and as reported previously.18 (link)Differential gene expression analyses and exploratory gene set enrichment analyses (GSEA) were performed after Robust Multi-array Average (RMA) normalization19 (link) and batch correction, with the Partek Genomics suite platform (v 6.6). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1–C7 version 5.0,20 (link) 1000 permutations and default additional parameters. An false discovery rate (FDR) threshold of 0.1 was applied.
3
Transcriptome Analysis of De Novo Glioblastoma
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Seventy-six fresh-frozen surgical samples of de novo GBMs were prospectively collected between 2010 and 2015. RNA was extracted with the Nucleospin® TriPrep (Macherey-Nagel, Düren, Germany) and the QIASymphony RNA (Qiagen, Venlo, The Netherlands) kits according to the manufacturers’ instructions. Affymetrix HG U133 plus 2.0 arrays were prepared and scanned according to the manufacturer’s protocol and as reported previously [54 (link)]. Quality control and differential gene expression analyses were performed with R (v3.2.2). Based on principal component analysis (PCA) plots, 3 outliers were removed. Robust Multi-array Average (RMA) normalization was applied. Batch correction was performed with the ‘sva’ package. Differential expression was analyzed with the ‘limma’ package and heatmaps were created with the ‘heatmap3′ package (R).
Exploratory Gene Set Enrichment Analyses (GSEA) were performed after RMA-normalization [55 (link)] and batch correction, with the Partek® Genomics suite platform (v6.6) (Partek, St. Louis, MO, USA). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1—C7 version 5.0 [56 (link)], 1000 permutations and default additional parameters. An FDR threshold of 0.25 was applied as recommended [55 (link)].
Exploratory Gene Set Enrichment Analyses (GSEA) were performed after RMA-normalization [55 (link)] and batch correction, with the Partek® Genomics suite platform (v6.6) (Partek, St. Louis, MO, USA). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1—C7 version 5.0 [56 (link)], 1000 permutations and default additional parameters. An FDR threshold of 0.25 was applied as recommended [55 (link)].
4
Quantifying Gene Expression in HEK 293T Cells
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GFP positive HEK 293T cells, transfected with either pEGFP or pEGFP-E1, were sorted by flow cytometry (BD FACSAria™ II, USA), and DNA, RNA, and protein from the sorted cells were extracted using NucleoSpin® TriPrep (Macherey-Nagel, Germany). Extracted RNA (3 μg) was reverse transcribed using Super Script IV (Invitrogen, USA) according to manufacturer protocol. The cDNA from pEGFP or pEGFP-E1 transfected cells was amplified by real-time PCR using PrimePCR™ 96 well as follows: 10μL of 2x SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, USA), 1μL of cDNA template, and 9μL of Nuclease-free H2O for a final volume of 20μL. The PCR reaction was as follows: initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds. GAPDH was used as a reference gene. Gene expression analysis was calculated using the 2-ΔΔCT method.
5
Isolation and Purification of Parasites
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Extraction was performed by following the methodology described by Lélu et al. [33 (link)]; briefly, to each 5 gr sample, 10 mL of deionized water was added; posteriorly, it was mixed for 1 minute using a vortex mixer. Then, 20 mL of Sheather's sugar solution (specific gravity: 1.2) was added and centrifuged at 1500 g for 20 minutes; the obtained interface (13 mL) was transferred to another tube in which 35 mL of deionized water was added and then centrifuged at 1500 g for 20 minutes. From each tube, 1 mL of sediment was collected and placed in a 1.7 mL Eppendorf tube; this was centrifuged at 1500 g for 5 minutes and, then, the supernatant was eliminated (approximately 600 μL), and the remaining sediment from each vial was then combined in one tube, resulting in one tube for each sample (superficial and deep) from their respective parks. Purification was made by using the NucleoSpin® TriPrep (MACHERY-NAGEL, Germany) kit and following its manufacturer recommendations.
6
DNA Extraction from Diverse Samples
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Apical tissue samples were homogenized in a lysis buffer with lysozyme 20 mg/mL at 37°C for 1 h. DNA was extracted using a commercial kit to obtain bacterial DNA (NucleoSpin® TriPrep, Macherey-Nagel), according to the recommendations of the manufacturer. Intracanal exudate DNA was extracted by the boiling modified method (Queipo-Ortuño et al., 2008 (link)); the Eppendorf tubes containing the paper points and the RTF medium were boiled in a water bath for 10 minutes, followed by cooling down to -20°C for 10 minutes and were finally centrifuged for five minutes at 1000 rpm. PBMC bacterial DNA was extracted in a lysis buffer using lysozyme followed by the TE buffer and DNEasy isolation kit (QIAGEN Inc., Valencia, CA, USA), according to the manufacturer. The concentration and quality of DNA were confirmed by the 260:280 ratio in a spectrophotometer (Bio-Tek, Winooski, VT).
7
Simultaneous RNA, DNA, and Protein Extraction from Brain Tissue
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Total RNA, DNA, and
protein isolation from brain tissue were performed using the NucleoSpinTriPrep
(Macherey-Nagel) mini kit for RNA, DNA, and protein purification.
Thus, it ensured the parallel isolation of RNA, DNA, and protein from
an undivided sample at a higher yield and purity.46 (link) For this purpose, the left hemispheres of mice brains were
ground to fine powders in the presence of liquid nitrogen using a
DEPC-treated mortar and pestle. Then cells are lysed by incubation
in a solution containing large amounts of chaotropic ions and applied
to the Triprep column. DNA, RNA, and protein were isolated separately
and sequentially according to the manufacturer’s instructions.
The obtained RNA and DNA quality were visualized in agarose gel electrophoresis,
and their concentrations were measured in the “NanoDrop”
device. After RNA isolation, cDNA synthesis was performed immediately
from the RNAs whose concentrations were calculated.
protein isolation from brain tissue were performed using the NucleoSpinTriPrep
(Macherey-Nagel) mini kit for RNA, DNA, and protein purification.
Thus, it ensured the parallel isolation of RNA, DNA, and protein from
an undivided sample at a higher yield and purity.46 (link) For this purpose, the left hemispheres of mice brains were
ground to fine powders in the presence of liquid nitrogen using a
DEPC-treated mortar and pestle. Then cells are lysed by incubation
in a solution containing large amounts of chaotropic ions and applied
to the Triprep column. DNA, RNA, and protein were isolated separately
and sequentially according to the manufacturer’s instructions.
The obtained RNA and DNA quality were visualized in agarose gel electrophoresis,
and their concentrations were measured in the “NanoDrop”
device. After RNA isolation, cDNA synthesis was performed immediately
from the RNAs whose concentrations were calculated.
8
Multiomics Analysis of Muscle Biopsies
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A portion of the biopsy was stored at 4°C in RNAlater™ Stabilization Solution (ThermoFisher Scientific Scientific). All biopsies were crushed using a cryo‐grinder, including a Cryolys cooling system (Bertin Instruments) and a Precellys 24 homogenizer (Bertin Instruments). Extractions of RNA, DNA, and proteins from the same sample were performed using the NucleoSpin® TriPrep (Macherey‐Nagel) kit. The muscle fibres were ground in the RP1 buffer provided with the kit, at 1 mL/10 mg fibre (with a maximum of ~25 mg biopsy), then lysed by adding β‐mercaptoethanol. DNA, RNA, and proteins were purified following the protocol provided by the manufacturer and stored at −80°C. Quantification of mitochondrial DNA and mRNA analyses were performed by qPCR and RT‐qPCR, respectively. The detailed procedure can be found in the supporting information . The primer sequences are listed in Tables S1 and S2 . Protein expression levels were determined by western blot. Experimental procedure and the list of primary antibodies used can be found in the supporting information .
9
Total RNA Extraction from Muscle
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The frozen powdered muscle sample was weighed, and subsequently, total RNA was extracted using a spin column DNA and RNA purification kit (NucleoSpin® TriPrep; Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. Nucleic acid concentration per milligram of muscle was determined using a spectrophotometer (Nanodrop 2000; Thermo Fisher Scientific, Waltham, MA, United States).
10
Quantitative PCR Analysis of GFP and GAPDH
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RNA was isolated from 1 × 106 NIH-HHD cells using the NucleoSpin TriPrep (MACHEREY-NAGEL). After RNA quality and integrity were verified, 2.8 μg of total RNA were used as template for cDNA synthesis with random hexamers, using SuperScript III Reverse Transcriptase (Invitrogen). Samples were diluted 1:5 and 5 μl used as template in a 20 μl qPCR reaction using SYBR Green PCR Master Mix (Thermo), with 500 nM primer concentration. Primer sequences: GFP: F:5′-acgacggcaactacaagacc -3′, R:5′-tgaagtcgatgcccttcag -3′; Gapdh: F:5′-tggagaaacctgccaagtatg -3′, R:5′-gttgaagtcgcaggagacaac -3′. Samples were run on the QuantStudio 3 Real-Time PCR system (Thermo Fisher) and analyzed according to the comparative ΔΔCt method.
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