Nucleic acid isolation from the collected biopsies followed a standard procedure. First, samples were weighted, and then 10–15 tumor cryosections (10–15 µm each) were prepared from each specimen. One section from the centre of each biopsy was stained with hematoxylin and eosin (H&E) and reviewed by an experienced pulmonary pathologist to determine the content of tumor cells vs. non-neoplastic cells, stroma and necrotic areas in the whole sample (18 (link)). Frozen sections of tumor were then homogenized with the TissueLyser mixer-mill disruptor (25 sec, 25 Hz, Qiagen, Hilden, Germany), followed by extraction of total RNA and genomic DNA using an AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA and DNA were quantified with the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, MA, USA), and RNA quality was assessed with the Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit (Agilent Technologies, Boeblingen, Germany).
Tissuelyser mixer mill disruptor
Manufactured by Qiagen
Sourced in Germany
The TissueLyser mixer-mill disruptor is a laboratory equipment designed to efficiently homogenize and disrupt a wide range of sample types, including tissue, plant, and other solid materials, for further processing and analysis. The device utilizes high-speed oscillation and grinding action to effectively break down samples into a fine, homogeneous powder or suspension.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Allprep dna rna mirna universal kit, by Qiagen (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Aprotinin, by Carl Roth (1 mentions)
N 20 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Allprep dna rna universal kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
5 protocols using tissuelyser mixer mill disruptor
1
Nucleic Acid Isolation from Tumor Biopsies
2
Glycodelin Detection in MPM Tissues
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For the detection of glycodelin in tissues of MPM patients, cryosections (10 - 15 mm thick) of snap frozen MPM tissues were prepared. For each 100 mg of tissue, 300 μl PBS with protease inhibitors (10 ng/ml Aprotinin, 100 μM Leupeptin, 1 μM Pepstatin, 1 mM PMSF, all Carl Roth, Karlsruhe, Germany) was added. Cryosections were homogenized with the TissueLyser mixer-mill disruptor (1 min, 25 Hz, Qiagen, Hilden, Germany) followed by a centrifugation step for 10 min with 13000 x g at 4°C. The supernatants of the samples were used for glycodelin detection by Western blot. Glycodelin was detected with the polyclonal N-20 antibody (sc-12289, Santa Cruz Biotechnology, Heidelberg, Germany). Anti-beta-actin (#A5441 Sigma-Aldrich) was used as a loading control.
3
Tumor Cell Isolation for Molecular Analysis
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Tissues were snap-frozen within 30 min after resection and stored at − 80 °C until the time of analysis. For nucleic acid isolation, 10 to 15 tissue cryosections (10 to 15 µm each) were prepared for each patient. The first and the last sections in each series were stained with hematoxylin and eosin (H&E) and tumor samples were reviewed by an experienced lung pathologist to determine the proportions of viable tumor cells, stromal cells, normal lung cell cells, infiltrating lymphocytes and necrotic areas. Only samples with a viable tumor content of ≥ 50% were used for subsequent analyses. Frozen tumor cryosections and matched normal lung tissue pieces were homogenized using the TissueLyser mixer-mill disruptor (2 × 2 min at 25 Hz, Qiagen, Hilden, Germany). Total DNA was isolated with the AllPrep DNA/RNA Universal Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA was stored at − 80 °C until further use.
4
Tumor and Tissue RNA Isolation and cDNA Synthesis
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Frozen tumor cryosections were homogenized with the TissueLyser mixer-mill disruptor (Qiagen). Matched normal lung tissue pieces were homogenized using a Miccra D-8 rotor-stator homogenizer (Art-moderne Labortechnik). Total RNA was isolated from tissue or from cultured cells using an RNeasy Mini Kit (Qiagen). The quantity of RNA was measured with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The quality of total RNA was assessed with an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies). Total RNA was transcribed to sscDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche) in three independent reactions that were afterwards pooled, mixed and stored at −80 °C until further analyses.
5
Protein Expression Analysis in Muscle
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Briefly, muscle samples were weighed and homogenised in a Tissue-Lyser mixer-mill disruptor (Qiagen) in treatment buffer, 19:1 w/v [0.125 mol/L Tris, 4% sodium dodecyl sulphate (SDS), 10% glycerol, 0.1 mol/L ethylenediaminetetraacetic acid and 5% β-mercaptoethanol]. Proteins from cultured cells were extracted using the same treatment buffer. Homogenised samples were loaded onto an SDS-polyacrylamide gel. Western blots were performed as described previously (Ref. 10) with minor modifications and probed with the following antibodies: melusin (Abcam), CD9 (R&D Systems), FRP-3 (anti-FRZB), GAPDH and ERK1/2 (Santa Cruz Biotechnology Inc), MyHC (Developmental Studies Hybridoma Bank), Integrin β1A and Integrin β1D (Millipore), Akt, P-Akt (Ser473), P-ERK1/2 (Thr202, Tyr204), GSK3β and P-GSK3β (Ser9) (Cell Signaling Technology).
Immunoreactive bands were visualised with the enhanced chemiluminescence reagent ECL-Plus (Amersham Pharmacia) and analysed with the Chemi-Doc XRS imaging system (Bio-Rad). The Student's t-test was applied to compare the mean values of different groups. Bars in graphs represent standard deviations.
Immunoreactive bands were visualised with the enhanced chemiluminescence reagent ECL-Plus (Amersham Pharmacia) and analysed with the Chemi-Doc XRS imaging system (Bio-Rad). The Student's t-test was applied to compare the mean values of different groups. Bars in graphs represent standard deviations.
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