For in vitro samples, cells were cultured as indicated and RNA was extracted using RNeasy mini kit (Qiagen) following manufacturer’s protocol. For tumor samples, tumors were dissected 2 weeks after injection. A 20 mg tissue from each sample was homogenized using Bead Ruptor 24 (Omni International) and RNA was processed using RNeasy mini kit following manufacturer’s protocol. Purified total RNA was cleaned up and mRNA was sequenced by NextSeq High Output. For analysis, reads were mapped to the mouse transcriptome (mm10, Ensembl annotations) using STAR (v2.7). FeatureCounts (v1.6) was used to count exonic reads (with -O option) and DESeq2 v1.20 (R v3.5.1) was used to normalize and compare samples. PCA plots were generated using the plotPCA function (DESeq2) after variance stabilization and dispersion estimation. Log-fold changes across all genes were used as input to iPAGE with the following parameters: ebins=9, max_p=0.005, and using MSigDB h gene sets (i.e. the Hallmarks gene set collection).
Bead ruptor 24
Manufactured by Omni International
Sourced in United States
The Bead Ruptor 24 is a high-speed bead mill homogenizer designed for the efficient disruption of a wide range of sample types. It utilizes a unique orbital shaking motion to effectively break down samples, enabling efficient extraction of target analytes. The Bead Ruptor 24 is capable of processing up to 24 samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
K3 edta tubes, by Sarstedt (2 mentions)
Silicon carbide sharp particles, by Biospec (2 mentions)
Omni br cryo cooling unit, by Omni International (2 mentions)
Br cryo cooling unit, by Omni International (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Nod scid gamma mice, by Jackson ImmunoResearch (2 mentions)
2.8 mm ceramic beads, by Omni International (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Omni International (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
1.4 mm ceramic beads, by Omni International (1 mentions)
Powerscanner microarray scanner, by Tecan (1 mentions)
Rneasy microarray tissue mini kit, by Qiagen (1 mentions)
Rna clean and concentrator 5 kit, by Zymo Research (1 mentions)
41 protocols using bead ruptor 24
1
mRNA Sequencing and Differential Gene Expression in Tumor Samples
2
Kinome Array Analysis of Frozen Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For kinome arrays, 40-mg tissue samples were flash frozen in liquid nitrogen and subsequently stored at −80°C until processing. Samples were thawed and homogenized using 1.4-mm ceramic beads (Omni International, Kennesaw, GA, USA) with lysis buffer (100 μl; Sigma-Aldrich, St. Louis, MO, USA) in a Bead Ruptor 24 (Omni International) for two 10-s cycles at machine speed 6. Homogenized tissue was spun in a microcentrifuge at 21,200 × g for 10 min at 4°C, and array protocols were carried out as previously described (46 (link)). After incubation for 2 h at 37°C, arrays were washed with shaking sequentially in phosphate-buffered saline (PBS) plus 1% Triton, twice in 2 M NaCl plus 1% Triton, and a final wash in distilled, deionized H2O. Arrays were scanned using a Tecan PowerScanner microarray scanner (Tecan Systems, San Jose, CA, USA) at 532 to 560 nm with a 580-nm filter to detect the phosphostain dye fluorescence. Images were gridded using GenePix Pro 7 software (San Jose, CA, USA), and spot intensity signal was collected. The data from GenePix were then analyzed by the PIIKA2 peptide array analysis software (47 (link)).
3
Brain RNA Extraction and Quality Assessment
4
Pharmacokinetics and Pharmacodynamics of RP-3500
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse whole blood was collected at 0.5, 1, 3, 8, and 24 hours postdose by tail snip and diluted 1:3 with 0.1 mol/L citrate buffer. Plasma was collected from whole blood drawn by cardiac puncture at 1 and 3 hours postdose into tripotassium (K3) EDTA tubes (Sarstedt; catalog no. 41.1504.105), then centrifuged at 12,000 relative centrifugal force for 10 minutes at 4°C. The concentration of RP-3500 in whole blood and plasma was determined by high-performance LC/MS (see Supplementary Methods). Excised tumors were flash-frozen for protein extract or preserved in 10% formalin. Frozen fragments were homogenized in lysis buffer (Mesoscale discovery, #R60TX-2) with protease and phosphatase inhibitors (Thermo Fisher Scientific, #78437; Thermo Fisher Scientific, #78420) using 2.8-mm ceramic bead-containing tubes (OMNI, #19–628) and a Bead Ruptor 24 (OMNI International). The levels of pCHK1(Ser345), total CHK1, pKAP1(Ser824), total KAP1, and pDNA-PKcs (Ser2056) were determined by immunoblotting relative to untreated controls as described above. The LoVo tumor IC80 for pCHK1(Ser345) was determined by a nonlinear least-squares regression to a normalized dose-response four-parameter fit model (GraphPad Prism v9).
5
Frozen Skeletal Muscle Metabolite Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A portion of the skeletal muscle biopsy samples were snap-frozen immediately after collection and stored at −80°C until the study was complete. The samples were batch-processed to minimize variation and were processed alongside a set of standard porcine muscle samples to assess the presence of experimental error. A more complete description of sample processing for the skeletal muscles is outlined elsewhere (17 ). Briefly, ∼15–25 mg frozen skeletal muscle was weighed and then transferred to a prechilled stainless steel homogenization tube loaded with silicon carbide sharp particles (Biospec Products). Prechilled 50:50 methanol:water extraction buffer was added to each tube at a ratio of 13 μL buffer/mg tissue and then a mixture of isotope-labeled standards for organic acids and acylcarnitines was added to each tube (see Supplemental Tables 1 2 for isotope standard details). Samples were then homogenized by using a Bead Ruptor 24 (Omni International, Inc.) with the use of two 30-s homogenization steps at 6.3 m/s with a subsequent dry ice cool-down step in between. Samples were centrifuged for 10 min at 14,000 × g at 4°C, and the raw extract supernatant was transferred to a fresh tube and stored at −80°C until subsequent analysis.
6
Tocopherol Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tocopherols were extracted and analyzed by the method suggested by Li et al. (2013) (link) with minor changes. About 100 mg of fresh leaf discs were placed in a 2-ml skirted screw-cap microtube having 1.3 ml of n-hexane. Samples were macerated using bead mill homogenizer (Bead Ruptor-24, Omni, Kennesaw, GA, United States) at 8 m s–1 for 30 s and incubated for 15 h under dark conditions. The supernatant (20 μl) was subjected to normal-phase high-performance liquid chromatography machine (model 600, Waters, Milford, MA, United States) fitted with a Zorbax Rx-SIL column (4.6 mm × 250 mm × 5 μm; Agilent, Englewood, CO, United States) and a fluorescence detector (λex = 295 nm; λem = 330 nm). The mobile phase of hexane/tert-butyl methyl ether (95:5, v/v) was transported at a constant rate of 1 ml min–1. Isoforms of tocopherol were quantified using the curves derived from pure standards of tocopherol. The peaks of the tocopherol standards (α-, β, γ-, and δ- tocopherol) were distinguished by their retention times. To determine tocopherol contents of experimental samples, standard curve was calibrated in accordance with the corresponding peaks of individual tocopherol derivatives. Tocopherol contents were expressed in mg per g of sample, and the total amount of tocopherol was calculated as the sum of α- and γ-tocopherol.
7
Pharmacokinetic Analysis of RP-3500 in Mice
8
Microbial Community DNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from 0.5 to 1.0 g of sediment from each sample using the DNeasy PowerLyzer PowerSoil Kit (12855-100, QIAGEN) according to manufacturer protocol with minor modifications for the step of homogenization and cell lysis, i.e., cells were lysed by bead beating at 6 m s−1 for 45 s using a Bead Ruptor 24 (OMNI). Extraction blanks were performed alongside the samples to assess laboratory contamination during the extraction process. DNA concentrations were assessed fluorometrically using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). The v3-4 region of the bacterial 16S rRNA gene and the v4-5 region of the archaeal 16S rRNA gene were amplified using the primer pairs SD-Bact-0341-bS17/SD-Bact-0785-aA21 and SD-Arch-0519-aS15/SD-Arch-0911-aA20, respectively (45 (link)), followed by post-PCR cleanup and indexing. Indexed amplicon samples were sequenced using Illumina’s v3 600-cycle (paired-end) reagent kit on an Illumina MiSeq benchtop sequencer (Illumina Inc.) after all DNA extraction blanks and PCR reagent blanks were confirmed for negative amplification. Raw sequences were quality controlled and further processed to construct an ASV table. Detailed methods for PCR conditions, amplicon cleanup, indexing, sequence processing, ASV taxonomy assignment, estimation of alpha and beta diversity, and associated statistical analyses are described in SI Appendix .
9
Quantifying Trabecular Bone mRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A sample of trabecular bone was taken from the distal right femur and homogenized in buffer provided by the manufacturer using a high-speed homogenizer (Bead Ruptor 24, Omni, Kennesaw, GA, USA) and steel beads at 4°C. The tissue lysate was incubated at 65°C and centrifuged to precipitate the debris.
mRNA expression was quantified using the QuantiGene Plex 2.0® technique (Panomics/Affymetrix Inc, Santa Clara, CA, USA) according to the manufacturer’s instructions. Oligonucleotide probe sets for the genes of interest were designed by the manufacturer. The tissue lysate was pipetted into a 96-well plate preloaded with capture reagent and a probe set. After incubation overnight at 54°C, hybridization with the preamplifier, amplifier, and biotinylated label was carried out. Luminescence was measured using a Luminex® instrument (Bio-Rad, Hercules, CA, USA), and the mean fluorescence intensity specific for each gene (proportional to the mRNA captured by the bead) was generated. Expression of each gene was normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase. The genes of interest are listed inTable 1 .
mRNA expression was quantified using the QuantiGene Plex 2.0® technique (Panomics/Affymetrix Inc, Santa Clara, CA, USA) according to the manufacturer’s instructions. Oligonucleotide probe sets for the genes of interest were designed by the manufacturer. The tissue lysate was pipetted into a 96-well plate preloaded with capture reagent and a probe set. After incubation overnight at 54°C, hybridization with the preamplifier, amplifier, and biotinylated label was carried out. Luminescence was measured using a Luminex® instrument (Bio-Rad, Hercules, CA, USA), and the mean fluorescence intensity specific for each gene (proportional to the mRNA captured by the bead) was generated. Expression of each gene was normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase. The genes of interest are listed in
10
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were pulverized using a Bead Ruptor 24 with an Omni BR-Cryo Cooling Unit (Omni International, Georgia, USA) and extracted in radio immunoprecipitation assay (RIPA) lysis buffer containing 1 mm PMSF. The homogenates were incubated for 0.5 h on ice to achieve full lysis and then centrifuged at 10,000 r·min-1 for 10 min at 4°C. The supernatants were transferred in aliquots to new tubes. The soluble proteins were quantified by a BCA protein assay kit. After being mixed with loading buffer and boiled for 10 min, the lysates were subjected to SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking, the immunoblots were incubated with the primary antibodies overnight at 4°C. After incubation with an HRP-linked secondary antibody, the blots were individually visualized using an enhanced chemiluminescence reagent kit. The bands were quantified by Quantity One software (Bio-Rad, Richmond, CA, USA) and normalized to GAPDH as an internal control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!