Tissuelyzer 2

Manufactured by Qiagen

Sourced in Germany, United States, Canada, Denmark

The TissueLyzer II is a laboratory equipment designed for rapid and efficient disruption and homogenization of samples, including tissue, plant, and bacterial samples. It utilizes a bead-beating technology to thoroughly break down the samples, preparing them for subsequent processing and analysis.

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215 protocols using tissuelyzer 2

Parenchymal Tissue Biopsy and DNA Extraction

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The fourth anterior mammary gland on the right side of the gilt was further dissected for the parenchymal or functional tissue. If the fourth mammary gland was not developed, the third anterior mammary gland was collected. A 12-mm biopsy punch was utilized over the teat area of the gland. Approximately 30 mg of tissue was taken from the tissue isolated from the biopsy punch for DNA extraction. After the biopsy punch was utilized, any remaining parenchymal tissue was dissected from the mammary gland (Figure 1). The parenchymal tissue was then weighed.
DNA extraction was then completed using a DNeasy Blood & Tissue kit (Qiagen, Germantown, MD) according to the manufacturer’s recommendations for “Purification of Total DNA from Animal Tissues” (Spin-Column Protocol). The entire process was performed at 20 °C, except where specified. Up to 30 mg of tissue was isolated for DNA extraction, and the tissue was disrupted using a Qiagen TissueLyzer II (Qiagen). The elution was quantified via a spectrophotometer (ND-100; Nanodrop Technologies, Inc., Rockland, DE) for DNA content, and all samples had a 260:280 ratio between 1.8 and 2.0 (Lucena-Aguilar et al., 2016 (link)).
The concentration of DNA was then used with the approximate 30 mg of tissue used for extraction and the 200 μL used in the final elution of DNA in the dissected parenchymal tissue using the following simplified equation:

Elefson S.K, & Greiner L.L. (2023). The evaluation of the supplementation of vitamin A, beta-carotene, and oxidized beta-carotene in prepubertal gilts. Journal of Animal Science, 101, skad103.

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Extraction and Storage of Cheetah Fecal Metabolites

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All fecal samples were dried, weighed, and extracted to the same final concentration. Frozen cheetah fecal samples were placed on ice. The lid and rim of each sample tube were cleaned with a Kimwipes (Kimberly-Clark) moistened with 70% ethanol (EtOH) to remove spurious fecal material. Samples were dried overnight using a Labconco CentriVac and either placed at –80°C for storage or immediately processed as follows. A clean spatula and tweezers were used to transfer 50 to 100 mg of dried stool into a new, labeled tube. Exact weights were recorded, and a 10-fold volume of cold 50% methanol (MeOH) in water was added to each tube (for example, 50 mg of stool plus 500 μl of 50% MeOH) (liquid chromatography-mass spectrometry [LC-MS]-grade solvents; Fisher Chemical). The samples were homogenized for 5 min at 25 Hz on a tissue homogenizer (Qiagen TissueLyzer II; Qiagen, Hilden, Germany) and subsequently placed at –20°C for 15 min for methanol extraction. The samples were then centrifuged at maximum speed (14,000 × g) for 15 min (Eppendorf US centrifuge 5418; USA). Without disrupting the pellet, 300 μl of the supernatant was transferred into a 96-deep-well plate. Samples were sealed and stored at –80°C.

Gauglitz J.M., Morton J.T., Tripathi A., Hansen S., Gaffney M., Carpenter C., Weldon K.C., Shah R., Parampil A., Fidgett A.L., Swafford A.D., Knight R, & Dorrestein P.C. (2020). Metabolome-Informed Microbiome Analysis Refines Metadata Classifications and Reveals Unexpected Medication Transfer in Captive Cheetahs. mSystems, 5(2), e00635-19.

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Liver RNA Extraction and Quantification

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Liver samples (5–20 mg) were homogenized in 700 μL QIAzol lysis reagent (Qiagen, # 79306) with Qiagen Tissuelyzer II (Qiagen, #85300) at 30 Hz for 2 min. Total RNA was extracted using miRNeasy Mini Kit (Qiagen, #217004) according to the manufacturer’s instructions. RNA quantity and quality were evaluated by Nanodrop™ 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific # ND-2000).

Behary J., Raposo A.E., Amorim N.M., Zheng H., Gong L., McGovern E., Chen J., Liu K., Beretov J., Theocharous C., Jackson M.T., Seet-Lee J., McCaughan G.W., El-Omar E.M, & Zekry A. (2021). Defining the temporal evolution of gut dysbiosis and inflammatory responses leading to hepatocellular carcinoma in Mdr2 −/− mouse model. BMC Microbiology, 21, 113.

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Liver Lipid Extraction Protocol

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Lipids were extracted from liver samples as previously described (Jain et al., 2022 (link)). Samples were kept on ice throughout the extraction. Briefly, liver was homogenized in 500 µl of 3:1:6 isopropanol:water:ethyl acetate containing internal standard in ceramic bead tubes (QIAGEN #13113-50) using the TissueLyzer II (QIAGEN #9244420). Samples were centrifuged at 16,000 × g for 10 min at 4°C and the lipid containing supernatant was transferred to a new 1.5 ml tube. Lipid extracts were dried in a SpeedVac (Thermo Savant RVT5105) and resuspended in 150 µl of methanol. Samples were kept at 4°C for no more than 1 week before analysis.

Varadharajan V., Ramachandiran I., Massey W.J., Jain R., Banerjee R., Horak A.J., McMullen M.R., Huang E., Bellar A., Lorkowski S.W., Gulshan K., Helsley R.N., James I., Pathak V., Dasarathy J., Welch N., Dasarathy S., Streem D., Reizes O., Allende D.S., Smith J.D., Simcox J., Nagy L.E, & Brown J.M. (2024). Membrane-bound O-acyltransferase 7 (MBOAT7) shapes lysosomal lipid homeostasis and function to control alcohol-associated liver injury. eLife, 12, RP92243.

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Protein Extraction and Analysis from Tissue Samples

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Skeletal muscle and tumor samples were prepared by using TissueLyzer II (QIAGEN) to homogenize samples in RIPA lysis and extraction buffer supplemented with protease inhibitors and phosphatase inhibitors (Sigma, St. Louis, MO) following the manufacturer’s instruction. The homogenate was incubated at 4°C for 2 h, and then centrifuged at 12000g, 4°C for 15 min. The supernatant was transferred into a fresh tube. Protein concentration was determined by Bradford Protein Assay. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA blocking buffer for 1 h and rinsed with TBST buffer. The membrane was incubated with primary antibody at 4°C overnight. After 3 washes with TBST buffer, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was washed with TBST buffer and then developed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and analyzed by Bio-Rad Gel Documentation System.

Li Y., Zhang F., Modrak S., Little A, & Zhang H. (2019). Chronic alcohol consumption enhances skeletal muscle wasting in mice bearing cachectic cancers: the role of TNFα/myostatin axis. Alcoholism, clinical and experimental research, 44(1), 66-77.

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Quantifying ELP1 Protein in Tissue Homogenates

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Tissue samples were collected, snap-frozen in liquid nitrogen, weighed, and homogenized on the TissueLyzer II (Qiagen) in RIPA buffer (Tris-HCl 50 mM, pH 7.4; NaCl 150 mM; NP-40 1%; sodium deoxycholate 0.5%; SDS 0.1%) containing a cocktail of protease inhibitors (Roche) at a tissue weight to RIPA buffer volume of 50 mg/mL. The samples were then centrifuged for 20 min at 14,000 × g in a microcentrifuge. The homogenates were transferred to a 96-well plate and were diluted in RIPA buffer to ~1 mg/mL for ELP1-HTRF and ~0.5 mg/mL for total protein measurement using the BCA protein assay (Pierce). Samples were run in duplicate and averaged. For the ELP1-HTRF assay, 35 μL of tissue homogenate were transferred to a 384-well plate containing 5 μL of the antibody solution (1:50 dilution of anti-ELP1 D2 and anti-ELP1 cryptate from Cisbio). The plate was incubated overnight at room temperature. Fluorescence was measured at 665 and 620 nm on an EnVision multilabel plate reader (Perkin Elmer). Total protein content was quantified in each tissue homogenate using the BCA assay according to the manufacturer’s protocol. The total protein normalized change in ELP1 protein signal for BPN-15477 and vehicle-treated tissue sample was calculated as ratio of the signal in the presence of the test compound over the signal in the absence of the test compound (vehicle control).

Gao D., Morini E., Salani M., Krauson A.J., Chekuri A., Sharma N., Ragavendran A., Erdin S., Logan E.M., Li W., Dakka A., Narasimhan J., Zhao X., Naryshkin N., Trotta C.R., Effenberger K.A., Woll M.G., Gabbeta V., Karp G., Yu Y., Johnson G., Paquette W.D., Cutting G.R., Talkowski M.E, & Slaugenhaupt S.A. (2021). A deep learning approach to identify gene targets of a therapeutic for human splicing disorders. Nature Communications, 12, 3332.

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RNA Extraction from Tissue Samples

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Total RNA was extracted from approximately 10 mg of minced tissue samples using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Initially, tissue samples were mixed with QIAzol reagent from the kit and then ground with a TissueLyzer II (Qiagen) using a 5-mm zirconia bead for 10 min at room temperature. RNA samples were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific) and their quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) with an RNA 6000 Nano total RNA Kit (Agilent Technologies).

Ohshima K., Hatakeyama K., Nagashima T., Watanabe Y., Kanto K., Doi Y., Ide T., Shimoda Y., Tanabe T., Ohnami S., Ohnami S., Serizawa M., Maruyama K., Akiyama Y., Urakami K., Kusuhara M., Mochizuki T, & Yamaguchi K. (2017). Integrated analysis of gene expression and copy number identified potential cancer driver genes with amplification-dependent overexpression in 1,454 solid tumors. Scientific Reports, 7, 641.

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Measuring Plant Biomass and Proteomics

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Aboveground (leaves + stems) and belowground (roots) fresh weight were manually recorded for ten replicates of each accession. Immediately after phenotypic data collection, the aboveground portion of each plant was harvested, weighed, and flash frozen in liquid nitrogen. Roots from each plant were washed and dried with paper towels, weighed and immediately flash frozen in liquid nitrogen. Of the ten flash-frozen replicates from each treatment and accession five were used for dry biomass measurements following lyophilization and five were saved for proteomics analysis. The lyophilized samples were ground using a Qiagen TissueLyzer II (Germantown, MD, USA) and sample powders were kept at −80 °C until metabolite extraction.

Handakumbura P.P., Stanfill B., Rivas-Ubach A., Fortin D., Vogel J.P, & Jansson C. (2019). Metabotyping as a Stopover in Genome-to-Phenome Mapping. Scientific Reports, 9, 1858.

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Mosquito RNA/DNA Extraction Protocol

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For RNA and DNA extraction, mosquito pools were processed as described by Scheuch et al. [40 (link)]. Briefly, samples were homogenized with three 3 mm steel beads in 450 µL (for single mosquitoes) or 750 µL (pools with more than one specimen) serum-free minimal essential medium, supplemented with penicillin, streptomycin, gentamicin and amphotericin B (ThermoFisher Scientific, MA, USA), for 2 min at 30 Hz in a Tissue Lyzer II (Qiagen, Hilden, Germany). Subsequently, debris was pelleted by centrifugation for 3 min at 20,000 g. 200 µL of the supernatant was heat-inactivated (10 min, 70 °C) before simultaneous DNA/RNA extraction according to the manufacturer’s instructions, using the NucleoMag Vet Kit (Macherey-Nagel, Düren, Germany) on a BioSprint 96 workstation (Qiagen). Extracted nucleic acids were used for RT-qPCR virus analysis, RT-PCR and sequencing, as well as for genetic mosquito species identification and biotype differentiation.

Kampen H., Holicki C.M., Ziegler U., Groschup M.H., Tews B.A, & Werner D. (2020). West Nile Virus Mosquito Vectors (Diptera: Culicidae) in Germany. Viruses, 12(5), 493.

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Bat Fly Processing for Marburg Virus Detection

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A total of 1653 bat flies collected from ERBs roosting at Matlapitsi cave between June 2012 and July 2018 were processed for laboratory testing. Bat flies were transferred into 2 mL grinding tubes and homogenised at 30 Hz for eight minutes using a Tissuelyzer II (Qiagen, Hilden, Germany) and 5 mm stainless steel beads (Qiagen). Cellular debris was removed by centrifugation at 14,000× g for 3 min, and the supernatant was used for virus isolation and detection of MARV RNA as previously described [28 (link),75 (link)].

Pawęska J.T., Jansen van Vuren P., Storm N., Markotter W, & Kemp A. (2021). Vector Competence of Eucampsipoda africana (Diptera: Nycteribiidae) for Marburg Virus Transmission in Rousettus aegyptiacus (Chiroptera: Pteropodidae). Viruses, 13(11), 2226.

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