RNA from fresh frozen primary tumors (D1, D2, and D3) was isolated using the Qiagen RNeasy kit (Qiagen, Hilden, Germany). Briefly, the tissue was placed in a 4.5 ml cryotube, and 500 µl of QIAzol (Qiagen) was added before the sample was disrupted using Qiagen TissueRuptor (Qiagen), according to the manufacturer’s recommendations. The sample was centrifuged at 18 400 ×g for 10 min to remove insoluble material before being processed with the Qiagen RNeasy kit with DNase. Samples were purified using the Zymo PCR inhibitor removal kit (Zymo, Irvine, CA). RNA from the pelleted samples (adherent and cultured spheres from D1, D2, and D3) was isolated as described above, except the disruption step using the Qiagen TissueRuptor. RNA concentration and purity were determined using NanoDrop (Wilmington, DE) and Bioanalyzer (Agilent 2100, Agilent, Santa Clara, CA). All nine samples had RNA integrity number (RIN) values above 8 before being analyzed with microarray and PCR [24 (link)].
Tissueruptor
Manufactured by Qiagen
Sourced in Germany, United States, Netherlands, United Kingdom, Australia, China, France, Japan, Spain
The TissueRuptor is a laboratory instrument designed for the disruption and homogenization of biological samples, such as tissues, cells, and other materials. It utilizes a high-speed rotor to efficiently break down the sample, enabling effective extraction of cellular components for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (33 mentions)
Trizol, by Thermo Fisher Scientific (33 mentions)
Nanodrop, by Thermo Fisher Scientific (28 mentions)
Trizol reagent, by Thermo Fisher Scientific (25 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (23 mentions)
Mirneasy mini kit, by Qiagen (21 mentions)
Qiazol lysis reagent, by Qiagen (19 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (18 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (17 mentions)
Rnalater, by Thermo Fisher Scientific (17 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Qiazol, by Qiagen (12 mentions)
Rneasy lipid tissue mini kit, by Qiagen (11 mentions)
Rneasy plus mini kit, by Qiagen (11 mentions)
2100 bioanalyzer, by Agilent Technologies (11 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (10 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Rneasy kit, by Qiagen (9 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Rneasy lipid tissue kit, by Qiagen (8 mentions)
441 protocols using tissueruptor
1
RNA Isolation from Fresh Frozen Tumors
2
Interscapular BAT RNA Extraction
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Mouse interscapular BAT was collected at time of euthanasia (~2pm) either in RNALater (Thermo Fisher Scientific) or snap-frozen, according to final use. For quantitative PCR (qPCR), total RNA was extracted from snap-frozen tissues using a TissueRuptor (Qiagen; Germantown, MD, USA) with disposable probes, and isolated using the EZNA total RNA kit I (Omega Biotek; Norcross, GA, USA). For microarray, total RNA was extracted from tissues in RNALater, processed with TissueRuptor and isolated using the Qiagen RNA extraction kit (Qiagen).
3
Tumor RNA Extraction and Quantification
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Tumor slices were disrupted with a TissueRuptor (QIAGEN) using TissueRuptor Disposable Probes for 15-20 s at maximum speed. RNA was then extracted from slices using AllPrep DNA/RNA Micro Kit (QIAGEN) and according the manufacturer’s protocol. Quality and quantity of RNA was assessed with Agilent RNA 6000 Pico or Nano kits (Agilent Technologies, Inc.) and NanoDrop ND-1000 spectrophotometer.
4
Bionano Prep Tissue DNA Isolation and DLE-1 Labeling
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DNA was extracted from tissue using the Bionano Prep SP Tissue and Tumor DNA Isolation Kit (Bionano, San Diego, USA) and a Tissue Ruptor (Quiagen) targeting a final concentration of 50–120 ng/ L. DLE-1 labeling was done using the Bionano Prep DLS Labeling Kit with final concentration targeted at 4–12 ng/ L. Data collection was performed using Saphyr 2nd generation instruments.
5
High Salt Extraction Protein Isolation
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High salt extraction (HSE) was preformed according to a previously described protocol [24 (link)]. Briefly, tissue samples from PC, DC, liver and small intestine, were homogenized in Triton X100 buffer using a tissue homogenizer (TissueRuptor, Quiagen, Hilden, Germany). The samples were centrifuged and the pellet was collected. The pellets were manually homogenized in high salt buffer containing 10 mM Tris-HCl, 140 mM NaCl, 1.5 M KCl, 5 mM EDTA, 0.5% Triton X100, 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA) and protease inhibitor mix (Complete, Roche, Mannheim, Germany), then mixed for 30 minutes at 4°C and re-pelleted. The pellets were washed in 5 mM EDTA/PBS buffer by manual homogenization, centrifuged to pellet, then re-suspended in Laemmli sample buffer. Samples were separated on a 10% acrylamide gel and stained using Coomassie Brilliant Blue.
6
Skin Biopsy RNA Extraction Protocol
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A 6-mm skin punch biopsy was obtained for each participant in the study. The homogenization of the tissue was carried out with the TissueRuptor (Quiagen) and the RNA was extracted with a RNeasy Plus Micro Kit (Quiagen), following the manufacturer’s instructions.
7
RNA Extraction from Liver and Soleus
8
Aortic Atherosclerosis Gene Expression
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Expression of 84 genes known to be involved in pathogenesis of atherosclerosis was examined in aortas of infected (n = 3) and control (n = 3) mice by qRT-PCR with the RT2 Profiler Mouse Atherosclerosis PCR Array (SABiosciences, Valencia, CA). Tissues were homogenized by QIAGEN TissueRuptor (QIAGEN, Valencia, CA). RNA was extracted using an RNeasy kit (QIAGEN), followed by reverse transcription with the RT2 First Strand Kit (QIAGEN). RNA samples were prepared for array with the SYBR Green Master mix (QIAGEN). Cycling was performed following manufacturer protocol, and data were analyzed using the PCR Array Data Analysis V4 excel worksheet, downloaded from the SABiosciences website.
9
Desmoglein 2 Protein Expression Analysis
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Primary tumors and mesenteric metastatic lesions were collected and homogenized in PBS with protease inhibitors (Roche, Mannheim, Germany) with a QIAGEN TissueRuptor (Qiagen). Homogenates were sonicated on ice for 30 seconds and then spun down at 4 °C at maximum speed in a table top centrifuge for 20 minutes. Samples in reducing Laemmli buffer were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis and blots were incubated with primary anti-human DSG2 (AbD Serotec, Raleigh, NC, 6D8, 1:1,000) and anti-human β-actin (Sigma) (1:3,000) antibodies and a horse radish peroxidase (HRP)-linked secondary anti-mouse IgG antibody (1:2,000).
10
Atherosclerosis Gene Expression in Mice
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Expression of 84 genes known to be involved in pathogenesis of atherosclerosis was examined in aortas of infected (n = 3) and control (n = 3) mice by qPCR with the RT2 Profiler Mouse Atherosclerosis PCR Array (SA Biosciences) [4 (link),5 (link)]. Tissues were homogenized by QIAGEN TissueRuptor (QIAGEN, Valencia, CA). RNA was extracted using an RNeasy kit (QIAGEN, Valencia, CA), followed by reverse transcription with the RT2 First Strand Kit (QIAGEN, Valencia, CA). Samples were prepared for array with the SYBR Green Master mix (QIAGEN, Valencia, CA). Cycling was performed following manufacturer’s protocol, and data was analyzed using the manufacturer’s PCR Array Data Analysis V4 excel worksheet [4 (link),5 (link)].
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