Fresh skeletal muscles were homogenized, employing the Precellys® 24 tissue homogenizer, and total RNA, enriched in miRNAs, was extracted using the mirVanaTM miRNA Isolation Kit (Invitrogen), according to manufacturer’s instructions. RNA was also extracted from exosomes using the Total Exosome RNA and Protein Isolation Kit (Invitrogen), following the manufacturer’s instructions. Reverse transcription of 10 ng from the RNA samples followed, using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Real-time PCR amplification was carried out with TaqMan MicroRNA Assays (Applied Biosystems) specific for miR-1, miR-133a, miR-133b, and miR-206 using the QuantStudioTM 7 Flex System (Applied Biosystems). Tissue and exosomal miRNA abundances were normalized to the endogenous control snoR-135 or spike-in control cel-miR-39, respectively.
Precellys 24
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Precellys 24 is a high-throughput tissue homogenizer designed for the efficient disruption and lysis of a wide range of sample types, including tissues, cells, and microorganisms. It utilizes bead-beating technology to effectively homogenize samples in a sealed tube, ensuring consistent and reproducible sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Precellys tube, by Bertin Technologies (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Mirvanatm mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Quantstudiotm 7 flex system, by Thermo Fisher Scientific (1 mentions)
Total exosome rna and protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Quant ittm ribogreen rna reagent and kit, by Thermo Fisher Scientific (1 mentions)
Diethylpyrocarbonate depc, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Intrizol reagent, by Thermo Fisher Scientific (1 mentions)
Abi prism 7000 cycler, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Qiagen (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Glycogen, by Merck Group (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Ribogreen rna quantification kit, by Thermo Fisher Scientific (1 mentions)
11 protocols using precellys 24
1
Quantification of Skeletal Muscle miRNAs
2
Optimized RNA Extraction and Integrity Verification
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Tissues were pulverized in a Precellys 24 homogenizer, and RNA was extracted with TRIzol (Invitrogen) following the modification of the method described by Chomczynski (1987) (link) based on the use of guanidine isothiocyanate followed by phenolic extraction (Chomzynski 1987 (link)). Total RNA was quantified by the Quant-iTTM RiboGreen RNA reagent and kit (Invitrogen, Life Technologies Corp.). Quality control of the extracted RNA was then performed in an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano Kit to verify the integrity of total RNA through band discrimination corresponding to fractions 18S and 28S of total RNA. All procedures involving RNA were performed with RNase-free tubes and filter tips and using water treated with diethylpyrocarbonate (DEPC, Sigma). The general RNA integrity number (RIN) obtained for analyzed samples is available in supplementary figure S11, Supplementary Material online.
3
RNA Extraction and Gene Expression Analysis
4
RNA Extraction and Reverse Transcription
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3–5 mm3 of frozen tissue from the left midfrontal region and temporal lobe was homogenised in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in a Precellys 24 homogenizer, incubated for 3 min in chloroform and centrifuged at 12,000g for 15 min at 4 °C. The upper aqueous phase was separated and mixed with an equal volume of isopropanol and 30 µg of glycogen (Sigma-Aldrich), incubated for 10 min, and centrifuged at 12,000g for 10 min at 4 °C for ribonucleic acid (RNA) precipitation. The RNA pellet was washed with 75 % ethanol, re-suspended in water (Sigma-Aldrich), and treated with DNase-I (40 U, Roche Diagnostics Ltd., West Sussex, UK) to remove genomic DNA. RNA concentration was measured using the Ribogreen RNA Quantification Kit (Invitrogen) and a FluoStar OPTIMA plate reader (BMG Labtech). RNA was reverse transcribed to complementary DNA (cDNA) using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA): 100 ng of RNA in a total volume of 100 µL was incubated at 25 °C for 10 min, 37 °C for 2 h, followed by inactivation at 85 °C for 5 s. cDNA concentration was determined using the Picogreen DNA quantification kit (Invitrogen) and FluoStar OPTIMA plate reader (BMG Labtech) according to the manufacturer’s instructions.
5
Measuring Renal Papillary cAMP Levels
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Blood pH was determined with an ABL90 Flex instrument (Radiometer GmbH, Krefeld, Germany). Mice were anesthetized with ketamine/xylazine (see above), blood was taken retroorbitally with a 65-µl capillary and blood parameters were measured according to manufacturer’s instructions. Then, mice were sacrificed by cervical dislocation and kidneys were removed. The papilla was prepared and collected in cAMP lysis buffer. After homogenization of the tissue with a Precellys® 24 homogenizer, the protein content of the papilla samples was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific). For cAMP concentration measurements, the AlphaScreen cAMP detection kit (Perkin Elmer) was used according to the manufacturer’s instructions. The relation between protein content and cAMP concentration of the samples was calculated to compare the samples of WT and KO papillae.
6
Macaque and Mangabey Fecal Sampling
7
Measurement of Prostaglandin Metabolites in Lice
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The Prostaglandin E Metabolite EIA Kit and the 13,14-dihydro-15-keto Prostaglandin F2α ELISA Kit (Cayman Chemical Company, Ann Arbor, MI, USA) were used to measure the levels of PGE2 and PGF2α metabolites in LsHPX1 KD copepodids and adult lice homogenates, according to supplier’s instructions with some modification as follows. For the PGE2 analysis, around 200 copepodids were pooled or one female was homogenized in 75 µl ddH2O using 1.4-mm zirconium oxide beads (Precellys 24; Thermo Fisher Scientific) and a TissueLyser LT (Qiagen) for 4 min at 50 Hz. Samples were centrifuged for 5 min at 1.5 g, and 55 µl of the supernatant was added to 16.5 µl carbonate buffer. After an overnight incubation on 37 °C, 22 µl of phosphate buffer and 16.5 µl EIA buffer were added, and 50 µl of the sample was added to two wells (two technical replicates). For the PGF2α analysis, around 150 copepodids were pooled in 100 µl ddH2O (N = 3) and homogenized as described above. Samples were centrifuged for 5 min at 1.5 g, and 50 µl of the supernatant was added to the ELISA wells. Two technical replicates were run for each of the three biological replicates.
8
Protein Expression Analysis of Tumor Cells
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Tumors and cells were homogenized in ice-cold RIPA buffer supplemented with a complete mini protease inhibitor cocktail tablet (Roche) using Precellys Ceramic Kit tubes in the Precellys 24 homogenizing instrument (Thermo Fisher Scientific). Proteins from lysates were separated on NuPAGE Novex Bis-Tris 10% gels (Invitrogen). Gels were transferred onto PVDF membranes using the iBlot Transfer system (Thermo Fisher Scientific). Membranes were probed with antibodies specific for ZEB1, N-cadherin, vimentin, HMGA2 (all as mentioned previously), N-MYC (Cell Signaling Technology Cat# 84406, RRID:AB_2800038), HMGCS (A-6, Santa Cruz Biotechnology), SQLE, LDLR (Proteintech Cat# 12544–1-AP, RRID:AB_2195888 and Proteintech Cat# 10785–1-AP, RRID:AB_2281164), Cleaved caspase-3 and cleaved PARP-1 (Cell Signaling Technology Cat# 9661, RRID:AB_2341188 and Cell Signaling Technology Cat# 5625, RRID:AB_10699459), or β-actin (Sigma-Aldrich Cat# A5441, RRID:AB_476744).
9
Macaque and Mangabey Fecal Sampling
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Frozen feces and intestinal tissue homogenates were accessed for this study. Approximately 1mL of macaque stool was originally collected fresh from each animal by inserting a sterile swab 2 cm into the rectum and spinning to collect available sample. Approximately 1 gram of mangabey feces was collected opportunistically from all animals within one hour of defecation on grossly uncontaminated surfaces (areas with no other pre-existing urine, water, food, natural substrates, or feces). Collected feces were snap-frozen and stored at −80°C until accession. To obtain intestinal biopsies, animals were sedated with 3-4 mg/kg Telazol administered intramuscular and isoflurane gas by intubation, to effect. Successful anesthetization was monitored by response to stimuli. For rectal biopsies, fecal material was removed from the rectum and biopsies obtained with biopsy forceps. Jejunal biopsies were obtained by video-guided endoscopy. 10 pinch biopsies were obtained per animal. Biopsies were maintained in RPMI-1640 medium for transport, rinsed twice with PBS, and transferred to soil-grinding Precellys tubes (Bertin Technologies, France). Samples were homogenized in 1 mL TRIzol (ThermoFisher Scientific, USA) at room temperature on a Precellys 24 homogenizer at 5000 revolutions per minute (RPM) in 4 successive 20 sec intervals and immediately transferred to −80°C for storage.
10
Tissue Homogenization for RNA Extraction
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Tissue samples were transferred while still frozen into screw-capped tubes containing approximately 8–10 1.4-mm zirconium oxide beads (Bertin Technologies KT03961-1-103.BK). Samples had appropriate amounts of TRIzol Reagent (Thermo Fisher #15596026) added as per manufacturer’s instructions. Samples were homogenized in Precellys 24 (Thermo Fisher) twice at 6000 rpm for 30 s with a hold period in between. The homogenized sample was transferred into an Eppendorf tube to remove the beads. The samples were then centrifuged 12,000×g for 5 min at 4 °C. The supernatant was collected in a new Eppendorf tube. Samples were stored at − 80 °C prior to nucleic acid extraction.
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