Grapevine leaves with P. viticola lesions were cut in small fragments (only the lesioned area with fresh mycelia and sporangiophores), submerged in sterile water (20 ml) and the lower surfaces were gently brushed to remove the emerging fungal mycelia and spores. The water suspension containing the fungal matrix was centrifuged at 5,800 g for 10 min. The resulting pellet was washed with sterile water (1 ml) and centrifuged at 14,000 g for 1 min. The pellet was finally resuspended in lysis buffer (Spectrum Plant Total RNA kit from Sigma-Aldrich) and 0.5 ml of glass beads (0.1 mm) were added in the same tube (video protocol: youtube.com/watch? v = 9ZMrUGRzMvw&t=). Two cycles of beadbeater (FastPrep-24 MP Biomedicals) at maximum speed for 30 s alternated with 2 min pauses were used to disrupt the fungal tissues. After RNA extraction following the RNA kit instructions (Spectrum Plant Total RNA kit from Sigma-Aldrich), sample concentrations were measured using a spectrophotometer (Nanodrop 2000, Thermo Scientific). An arbitrary quantity threshold was set of 50 ng/µl and only 141 RNAs passed the check (74 from Italy and 67 from Spain). Selected samples were then pooled to have the same final concentration (7 ng/µl; Supplementary Table S1 ), resulting in seven pools from Italy and nine pools from Spain.
Mp fastprep 24
Manufactured by MP Biomedicals
Sourced in United States
The MP FastPrep-24 is a high-speed benchtop homogenizer designed for the rapid and efficient disruption of a wide range of sample types, including tissues, cells, and microorganisms. The instrument utilizes a linear reciprocating motion to agitate samples with ceramic beads, effectively breaking down the sample matrix and releasing the desired analytes.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Lysing matrix d, by MP Biomedicals (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Qiaamp fast dna stool mini kit, by Qiagen (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Rna free dnase, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Salmon sperm dna, by Merck Group (2 mentions)
Oligo dt primer, by Thermo Fisher Scientific (2 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Rnaex total rna isolation solution, by Generay (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
33 protocols using mp fastprep 24
1
Extraction and Quantification of Fungal RNA
2
Faecal Metabolome Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freeze-dried faecal samples were added to the extraction solvent and the cells were disrupted using bead beating (FastPrep 24 MP Biomedicals), sonication, and freeze-thaw lysis methods. The samples were then centrifuged at 13,000× g for 15 min and the supernatant recovered. The samples were evaporated to dryness using a SpeedVac Savant SPD121P system (Thermo Scientific, Milford, UK) and stored at −80 °C until further processing. Reconstitution was performed in 250 μL 50/50 H2O: acetonitrile (ACN), vortexed for 1 min and centrifuged at 15,000× g for 15 min, and aliquots transferred into glass vials for MS analysis. Quality control (QC) samples were prepared by pooling samples across all groups undergoing simultaneous analysis. Solvent blanks and QC samples were entered at the beginning of every analytical run, and after every five samples in each batch over the course of the study to assess background in the system and detect potential contaminations. Experimental details for each extraction parameter are shown (Table 1 ).
3
Total RNA Extraction from Aurantiochytrium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA of Aurantiochytrium sp. PKU#SW7 was extracted with RNAExTM total RNA isolation solution (Generay, Shanghai, China) according to the manufacturer’s guidelines. Briefly, samples were homogenized in 1 mL RNAExTM using a MP homogenizer (MP FastPrep®-24, Santa Ana, CA, USA). Then, 200 μL of chloroform was added and the mixtures were incubated for 2 min at room temperature. After centrifugation at 12,000 g for 10 min, the aqueous phase was transferred to a fresh tube, and 500 μL of isopropyl alcohol was added for RNA precipitation and recovery through centrifugation at 12,000 g for 10 min. Afterwards, 1 mL of 75% ethanol was added for RNA washing and the RNA pellet was dissolved in DEPC (diethyl pyrocarbonate) treated water. The integrity of RNA was detected by agarose gel electrophoresis and the concentration was estimated by a UV-Vis spectrophotometer Nanophotometer P300 (Implen, Munich, Germany) and Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). RNA samples with OD260/280 ≥ 1.8, OD260/230 ≥ 1.8, and RIN > 9.5 were selected for cDNA library construction. Finally, the cDNA libraries were sequenced at Beijing Genomic Institute (BGI)-Shenzhen, China, using Illumina HiseqTM 2000 according to the manufacturer’s instructions (Illumina, San Diego, CA, USA).
4
Muscle Protein Extraction and Mitochondria Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aliquots of biceps femoris muscle were diluted in 400 μL lysis buffer (100 mmol/L NaCl, 20% (V/V) NP‐40, 20 mmol/L Tris‐HCl pH 7.4, 5 mmol/L MgCl2, 100 mmol/L PMSF) and mechanically disrupted in MP FastPrep®‐24 (M.P. Biomedicals, Irvine, CA). Hereafter, the samples were kept on ice and vortexed every ten minutes followed by the first centrifugation (30 min, 14,000g, 4°C). Supernatant was transferred into a new tube and 500 μL of lysis buffer was added. Vortexing and centrifugation (30 min, 14,000g, 4°C) were repeated and the supernatant, containing the whole cell protein lysate, was separated.
For isolation of intact mitochondria, quadriceps femoris muscle was directly processed after preparation, using the protocol described by Gostimskaya and Galkin (2010 ). Protein content was determined photometrically using Pierce® BCA Protein Assay Kit (Thermo Scientific Inc., Waltham, MA).
For isolation of intact mitochondria, quadriceps femoris muscle was directly processed after preparation, using the protocol described by Gostimskaya and Galkin (
5
Quantifying α-Synuclein in Neurological Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Roughly 50 mg of NHP brain tissue was added to 1 mL of RIPA buffer (Boston Bioproducts) with protease and phosphatase inhibitor tablets (Sigma-Aldrich) in a 2 mL Lysing Matrix D tube (MP Biomedicals). The samples were homogenized using an MP Fastprep-24 (MP Biomedicals), and total protein was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific) and normalized to 1 mg/mL. Tissue samples were diluted either 1:100 (spinal cord), 1:2000 (brain tissue) or 1:10 (CSF) prior to aSyn protein determination. aSyn protein concentrations were measured using the LEGENDMAX Human Alpha Synuclein ELISA Kit (BioLegend) following the manufacturer’s protocol. CSF hemoglobin levels were also analyzed using the human hemoglobin ELISA kit (Bethyl Laboratories). This was done to assess the impact of blood contamination on the aSyn levels detected in CSF due to the high levels of aSyn contained in RBCs. Samples with greater than 1000 ng/mL HgB were excluded from the CSF analysis.
6
Protein Extraction and Separation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To the lysis samples, the SDT buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) was added to the liver tissues, and an Automated Homogenizer (MP Fastprep-24, 6.0M/S, 30S) was used to homogenize the lysate twice. Boiling, centrifugation, and filtration were used to extract the homogenate supernatant. The amount of protein was quantified as previously described (23 (link)). The protein extracts were digested with trypsin based on a filter-aided sample preparation (FASP) procedure (24 (link)). Next, 12.5% SDS-PAGE was used to separate the proteins, and Coomassie Blue R-250 staining was used to visualize the protein bands (25 (link)).
7
Anorectal Epithelial Cell RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The colon, including the anorectal section, was opened longitudinally and cleaned from feces and fat tissue. Epithelial cells were harvested by incubating the tissue for 45 min in Hank’s Balanced Salt Solution (HBSS) supplemented with 5 mM EDTA and 10 mM HEPES at 37 °C. Cells were pelleted and resuspended in 500 μl Trizol (Life Technologies) and macerated using MP FastPrep-24 (MP Biomedicals). RNA was extracted according to the ImmGen protocol (Trizol.pdf">https://www.immgen.org/Protocols/Total%20RNA%20Extraction%20with%20Trizol.pdf ) and cDNA was transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed using qPCR SYBR Green Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Primer sequences are in Supplementary Information Table 1 .
8
Quantitative Analysis of Viral and Immune Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from PBMC, BAL samples, and frozen lung tissue using the QIAamp DNA Mini Kit (QIAGEN). RNA extraction was performed by homogenizing pooled frozen lung biopsies (n = 3 per animal) in 1 mL TRIzol reagent (Invitrogen) using the MP Fastprep-24 (MP Biomedicals) and the RNeasy Mini Kit (QIAGEN). cDNA synthesis was performed using 5 μg total RNA and Superscript IV reverse transcriptase (Invitrogen) with oligo(dT) primers (Invitrogen). qPCR was performed in duplicate on an ABI Prism 7500 using TaqMan 2× Universal Master Mix (Applied Biosystems). DNA qPCR was performed with primers and probes specific for SVV ORF21 and the single copy gene OSM (70 (link)) (Supplemental Table 2 ). cDNA qPCR was performed with primers and probes specific for SVV ORF63 (17 (link)) (Supplemental Table 2 ), OSM (70 (link)), CXCL10 (Mf02788358_g1) (47 (link)), CDH5 (Mf00901470_m1), CLDN2 (Mf04369164_m1), CLDN10 (MF02862371_m1), CLDN18 (Mf02805373_m1), DDX58 (Mf02789247_m1), GAPDH (Mf04392546_g1), IFIT1 (Mf04355804_m1), IFN-α2 (Mf04256335_s1), IFN-γ (Mf02788577_m1), MX1 (Mf00895608_m1), and STAT1 (Hs01013996_m1) (Applied Biosystems).
9
Genomic DNA Extraction from Plant Shoots
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From the stem tip, five 5- to 8-cm long shoots were arbitrarily selected from each of the total 96 replicate samples. For each shoot, ‘Vardar Valley’ had an average of 20 leaves, and ‘Justin Brouwers’ had an average of 40 leaves. We used liquid nitrogen to homogenize the shoot samples. The extraction of genomic DNA followed Qiagen Plant Mini Kit (Hilden, Germany) with minor modifications. Specifically, approximately 200 mg ground tissue was further homogenized using a MP FastPrep™ 24 (MP Bio, Irvine, CA, USA) at the speed of 4/s for 1 min in a sterilized zirconium bead tube (500 um garnet and 6 mm zirconium, PFMM 500-100-25U, OPS Diagnostics, Lebanon, NJ, USA) prefilled with 400 µl of AP1 buffer. Four µl of RNase A was added and vortexed for 3 s. Nuclease free water was used to dissolve DNA from the MB Spin Column membrane. The DNA was stored at -20°C.
10
Screening for Antibiotic Biosynthesis Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The same isolates that were taxonomically characterized were also tested for the presence of genes involved in the biosynthesis of antibiotics: the phlD gene for the production of DAPG, the phz gene for PCA, the pltB gene for pyoluteorin, and the prnC gene for pyrrolnitrin. A 2-mL sample of bacteria grown in Standard I Nutrient Broth (MERCK, Darmstadt, Germany) for 24 h at 25°C was centrifuged (14.400 rpm for 5 min at 4°C). The supernatant was discarded, the cells in the pellet were disrupted by means of a High-speed benchtop homogenizer MP FastPrep 24 (MP Biomedicals Germany GmbH, Eschwege, Germany), and DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instruction for use. The phlD, phz, pltB, and prnC genes were detected by quantitative real-time PCR (qPCR) with the primer pairs B2BF/BPR4, PCA2a/PCA3b, PltBf/PltBr, and Prncf/Prncr, respectively, described by Shirzad et al. (2012) (link). The PCR product size and purity were checked using the melting profile and agarose gel electrophoresis.
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!