Tissuelyser 2

Manufactured by Qiagen

Sourced in Germany, United States, United Kingdom, Netherlands, Japan, Italy, France, Australia, Switzerland, Canada, Denmark, China, Spain, Sweden, Belgium, Finland

The TissueLyser II is a laboratory equipment designed for efficient homogenization and disruption of biological samples, such as tissue, plants, and microorganisms. It utilizes a bead-milling technique to rapidly and thoroughly break down samples prior to further processing and analysis.

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2 632 protocols using tissuelyser 2

Standardized Stool and Tissue DNA Extraction

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Only one stool sample (200 mg approximately) was homogenized for each location. Each sample was homogenized twice in a 2 ml Eppendorf safe-lock containing 1 ml of sterile PBS and one steel ball of 5 mm (Werfen Ref.: BAI5-1000) for 2 min at 30 frequency with the Tissue Lyser II (QUIAGEN, Hilden, Germany) . Later, a first centrifugation step was performed to discard excessive non-degradable matter at 10,000×g for 30 s (standard rotor F45-24-11, Eppendorf centrifuge 5415R). The supernatant was collected and added to 900 μl of fresh PBS and vortexed. A second centrifugation was carried out at 4000×g for 15 min to obtain the pellet for DNA extraction. The pellet was re-suspended in μl of BT1 buffer with 25 μl of proteinase K and incubated overnight at 56 • C for DNA isolation.
A piece of tail tissue (0.5 cm) was homogenized in 180 μl of BT1 buffer with 25 μl of proteinase K in a 2 ml Eppendorf safe-lock containing and one steel ball of 5 mm for 2 min at 30 frequency with the Tissue Lyser II (QUIAGEN) and incubated overnight at 56 • C. After a centrifugation step at 11,000×g for 5 min, 200 f μl of supernatant were used for lysis and DNA isolation.
DNA isolation was carried out with the SPEEDTOOLS DNA extraction kit (BIOTOOLS, Madrid, Spain) according to the manufacturer's instructions. DNA yield of all samples was 10-500 ng/μl with 260 and nm UV absorbance ratios (A260/280) of 1.9-2.1.

Bermejo-Nogales A., Rodríguez Martín J.A., Coll J, & Navas J.M. (2022). VKORC1 single nucleotide polymorphisms in rodents in Spain. Chemosphere, 308(Pt 1).

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Testis and Epididymis Protein Extraction

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Testis and epididymides were dissected from wild-type and Tcp11^−/− adults and placed into PBS. Testis samples were lysed in 1 mL RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, 1% Nonident P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors) using Qiagen’s TissueLyser II (30 Hz, 2 min, RT). Epididymides were first minced with dissecting scissors in 1 mL RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT, 1% Nonident P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors) and then lysed with Qiagen’s TissueLyser II (30 Hz, 2 min, RT). Lysates were clarified with a 10 min spin at 18 000×g at 4 °C. The protein concentration was determined with the Bradford method. Thirty microgram per sample were loaded per well. Western blot analysis was performed using BioRad’s TransBlot Turbo. PVDF membranes were blocked with TBS with 0.05% Tween and 5% milk and washed with TBS with 0.05% Tween and 0.5% milk. Both primary and secondary antibodies were diluted in washing buffer. Nacalai’s Super Signal was used for chemiluminescence and detected with Image Quant. Antibodies used include the following: goat anti-BASAGIN 1:1000 (Santa Cruz), mouse anti-AKAP4 1:1000 (BD Biosciences), and mouse anti-Acetylated Tubulin 11 000 (Sigma).

Castaneda J.M., Miyata H., Archambeault D.R., Satouh Y., Yu Z., Ikawa M, & Matzuk M.M. (2019). Mouse t-complex protein 11 is important for progressive motility in sperm. Biology of Reproduction, 102(4), 852-862.

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Quantifying Parabacteroides copri in Mouse Feces

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P. copri DSM18205 bacteria were harvested from agar plates with sterile loops, washed by suspending in 0.9% NaCl and spinning down, resuspended in 0,9% NaCl and a fraction was plated out for colony counting. For the pure P. copri sample, a volume corresponding to 10⁹ bacteria was centrifuged at 3000 rpm and resuspended in lysis buffer. For the P. copri-spiked murine faeces samples, faeces were collected from C57BL/6 J mice (Janvier), homogenized using a Qiagen Tissuelyser II and aliquoted. Each aliquot was spiked with 10 uL of up to eight 10-fold dilutions of P. copri bacteria in 0.9% NaCl with 10¹⁻ 10⁹ CFU/mL. gDNA from bacteria only or faeces spiked with different amounts of bacteria was then prepared according to the manufacturor’s instructions using the QIAamp PowerFecal DNA kit (Qiagen, Hilden, Germany). Samples were added to lysis buffer and incubated at 65 °C for 10 min and disrupted and homogenized in a Bead Tube containing garnet beads, using a Tissuelyser II (Qiagen) prior to DNA extraction. DNA concentrations and purity were measured using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).

Verbrugghe P., Van Aken O., Hållenius F, & Nilsson A. (2021). Development of a real-time quantitative PCR method for detection and quantification of Prevotella copri. BMC Microbiology, 21, 23.

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RNA Extraction from Rice and Arabidopsis

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Two weeks after seeding, the youngest fully expanded leaves of O. sativa were frozen in liquid nitrogen and stored at -80 °C prior to RNA isolation for RNA-Seq. Frozen samples were homogenized with zirconia beads YTZ-4 (AS-ONE, Osaka, Japan) using TissueLyser II (Qiagen, Hilden, Germany), and total RNA was extracted using the Maxwell 16 LEV Plant RNA Kit (Promega, Madison, WI, USA) and the Maxwell 16 Automated Purification System (Promega). The concentration of RNA was measured using a QuantiFluor RNA System (Promega) and Quantus Fluorometer (Promega, Madison, WI, USA).
Seven days after seeding, bulked seedlings of A. thaliana were homogenized with zirconia beads YTZ-4 using a TissueLyser II (Qiagen, Hilden, Germany), and total RNA was extracted using the Maxwell 16 LEV Plant RNA Kit (Promega, Madison, WI, USA) and the Maxwell 16 Automated Purification System (Promega, Madison, WI, USA). The RNA concentration was measured using a Quantus Fluorometer (Promega, Madison, WI, USA).

Kashima M., Kamitani M., Nomura Y., Mori-Moriyama N., Betsuyaku S., Hirata H, & Nagano A.J. (2022). DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate. Plant Methods, 18, 99.

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Viral RNA Extraction and Detection in Organ Samples

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Organ samples were homogenized in 1 mL of sterile PBS using the Tissue Lyser II (Qiagen, Hilden, Germany) with 5 mm steel balls. The sample material was eluted from the swab heads in 1 mL of sterile PBS using the Tissue Lyser II (Qiagen). RNA was extracted with the cador Pathogen 96 QIAmp HT Liquid kit (Qiagen) using the QIAcube HT device, according to the manufacturer’s instructions. A four-microliter RNA extract was used as the template for the one-step RT-PCR (AIV qPCR TaqMan Fast Virus 1-Step Master Mix; Thermo Fisher Scientific Inc., Waltham, MA, USA). The primers and probe used for the amplification and detection of a fragment of the matrix gene were described previously [22 (link)].
The cutoff value of the method was set to Ct 36; i.e., samples with Ct < 36 were considered as positive and ≥36 as negative. The negative samples with no amplification were set to the maximum cycle number of 40.

Bóna M., Földi J., Dénes L., Harnos A., Paszerbovics B, & Mándoki M. (2023). Evaluation of the Virulence of Low Pathogenic H9N2 Avian Influenza Virus Strains in Broiler Chickens. Veterinary Sciences, 10(12), 671.

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Fecal DNA Extraction for Diverse Species

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DNA extraction was performed following the manufacturer’s recommendations with small adaptions due to equipment availability. Briefly, for cat, dog, and horse samples 25 mg of feces was placed into a 2 mL sterile round-bottom tube containing 500 μL of sterile PBS and a 0.5 cm diameter stainless steel bead. Samples were mechanically disrupted using a TissueLyser II (Qiagen, Venlo, Netherlands) for 2 minutes at 25 Hz, followed by centrifugation at 14,000 × g for 2 minutes. A 400 μL volume of supernatant was removed and placed in a sterile 2 mL screw cap tube containing 2 mg of sterile glass beads and 100 μL of lysis buffer ATL. Samples were processed on a TissueLyser II (Qiagen, Venlo, Netherlands) for 10 min. at 50 Hz and the remainder of the protocol was followed as recommended by the manufacturer. For mouse samples, one fecal pellet was placed into a 2 mL sterile round-bottom tube containing 500 μL of sterile PBS and a 0.5 cm diameter stainless steel bead and the sample was processed as described above. For fish samples, the entire GI tract was placed in a 2 mL sterile round-bottom tube containing 500 μL of sterile PBS and a 0.5 cm diameter stainless steel bead and the sample was processed as described above. All samples were eluted in 150 μL AE buffer.

Hart M.L., Meyer A., Johnson P.J, & Ericsson A.C. (2015). Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing. PLoS ONE, 10(11), e0143334.

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RNA Isolation from Cell Cultures and Tissues

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For RT-qPCR analyses, total RNA was isolated directly from culture cells using the NucleoSpin™ RNA Columns (Macherey-Nagel™, DE; cat# 740955) according to the instructions of the manufacturer. Mice tissues were disrupted using a Qiagen Tissuelyser II (as previously described) after the skin was accurately removed. Total RNA from the ESTDAB cells #002, #033, #039, #043, #047, #067, #095, #097, #114, #115, #128, #129, #135, #160, #161, #162, #199 was obtained using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, DE; cat# 80004) according to the instructions. For RNA-seq analyses, RNA from tissues (disrupted with Qiagen Tissuelyser II) were isolated using the RNeasy Plus Mini Kit (Qiagen, DE; cat# 74134), following the instructions of the manufacturer. When RNA from tissues could not be isolated at the moment, tumor pieces were preserved in RNAlater™ Stabilization Solution (ThermoFisher Scientific, MA, USA; cat# AM7020), according to the instructions.

Di Leo L., Bodemeyer V., Bosisio F.M., Claps G., Carretta M., Rizza S., Faienza F., Frias A., Khan S., Bordi M., Pacheco M.P., Di Martino J., Bravo-Cordero J.J., Daniel C.J., Sears R.C., Donia M., Madsen D.H., Guldberg P., Filomeni G., Sauter T., Robert C., De Zio D, & Cecconi F. (2021). Loss of Ambra1 promotes melanoma growth and invasion. Nature Communications, 12, 2550.

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Multiomic Analysis of Tissue Samples

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Total RNA was extracted using TRIzol reagent. Isolated tissues were homogenized using the Qiagen TissueLyser II in TRIzol to isolate total RNA and processed according to the manufacturer's instruction. Total RNA (1ug) was reverse transcribed to cDNA using the qScript cDNA Synthesis kit. RT-qPCR was performed using the CFX384 Real-Time PCR System using SYBR Green. For protein analyses, tissues were homogenized with the Qiagen TissueLyser II in 2% SDS lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured by bicinchoninic protein assay. Proteins were then resolved on 12% SDS-PAGE. The protein gels were then transferred to polyvinylidene fluoride (PVDF) membranes. Blots were probed with target antibodies and visualized using the FluorChem imaging system. Images were quantified with densitometry using the AlphaView software.

Liu Y., Qu Y., Cheng C., Tsai P.Y., Edwards K., Xue S., Pandit S., Eguchi S., Sanghera N, & Barrow J.J. (2023). Nipsnap1—A regulatory factor required for long-term maintenance of non-shivering thermogenesis. Molecular Metabolism, 75, 101770.

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RNA and Protein Extraction from Breast Tumor

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For RNA extraction, breast tumor samples were homogenized in RLT buffer (Qiagen, Valencia, CA, United States) supplemented with 1% 2-mercaptoethanol (Thermo Fisher Scientific) using a TissueLyser II (Qiagen) at a frequency of 30/s for 5 min. Homogenized tissue were resuspended in TRIzol™ Reagent (Thermo Fisher Scientific) and stored at −80°C until use. Sorted CD4⁺ T cells for RNA sequencing were resuspended in RLT buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Thermo Fisher Scientific) and stored at −80°C until use. RNA was isolated using the RNeasy Mini Kit (Qiagen), quantified using a NanoDrop ND-1000 spectrophotometer, and stored at −80°C until use.
For protein extraction, breast tumor samples were homogenized in phosphate-buffered saline (PBS) supplemented with 0.1% v/v Tween 20 (Sigma-Aldrich) and 4% protease inhibitor (Thermo Fisher Scientific) using a TissueLyser II (Qiagen) at a frequency of 30/s for 5 min. Samples were transferred to new tubes, frozen in liquid nitrogen for 1 min, and then thawed in a 37°C water bath for 3 min. Subsequently, samples were sonicated for 1 min, followed by centrifugation at 13,300 rpm for 10 min at 4°C. The aqueous phases containing protein extract was transferred to a new tube and stored at −80°C until use.

Boieri M., Marchese E., Pham Q.M., Azin M., Steidl L.E., Malishkevich A, & Demehri S. (2022). Thymic stromal lymphopoietin-stimulated CD4+ T cells induce senescence in advanced breast cancer. Frontiers in Cell and Developmental Biology, 10, 1002692.

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RNA Extraction from Adult and Fetal Hearts

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For adult hearts, whole ventricles were dissected and frozen in TRIzol immediately after dissection. The hearts were homogenized at 30 kHz for 1 min using TissueLyser II (Qiagen). The homogenized samples were centrifuged to pellet insoluble tissue. A Direct-zol RNA MiniPrep kit (Zymo Research) was used to isolate DNA-free RNA.
To isolate RNA from CPs, the sorted cells were lysed in TRIzol and RNA was extracted using phenol-chloroform.
To isolate RNA from human fetal tissue, whole hearts were stored in RNAlater Stabilization Solution (Thermo Fisher Scientific) and frozen immediately after isolation. The hearts were later thawed and the ventricles were dissected away from the atria and major vessels. The ventricles were cut open and rinsed for 10 s in PBS to remove blood inside the chamber and then immediately placed in TRIzol. The ventricles were homogenized at 30 kHz for 1 min using TissueLyser II (Qiagen). Homogenized samples were centrifuged to pellet insoluble tissue. A Direct-zol RNA MiniPrep kit (Zymo Research) was used to isolate DNA-free RNA.

Ahmed A., Liang M., Chi L., Zhou Y.Q., Sled J.G., Wilson M.D, & Delgado-Olguín P. (2020). Maternal obesity persistently alters cardiac progenitor gene expression and programs adult-onset heart disease susceptibility. Molecular Metabolism, 43, 101116.

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