Mosquitoes with at least three legs were selected for the MALDI-TOF MS analysis. Each mosquito was treated individually by grinding the legs in a 1.5-ml Eppendorf tube containing 15 μl of 70% (v/v) formic acid (Sigma-Aldrich, St. Louis, MO, USA) and 15 μl of 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland), with 1.0-mm-diameter glass beads (Sigma France, Lyon, France) using a tissue lyser machine (Qiagen); the optimal parameters had been previously established [36 (link)]. The crushed legs were centrifuged, and a 1-μl aliquot of the supernatant of each sample was deposited in quadruplicate on a MALDI-TOF MS steel plate (Bruker Daltonics, Wissembourg, France) and covered after drying with a matrix solution composed of 1 μl of saturated alpha-cyano-4-hydroxycinnamic acid (Sigma France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Sigma-Aldrich Co. Ltd., Gillingham Dorset, UK) and high-performance liquid chromatography-grade water [19 (link), 37 (link)]. The target plate was then air-dried for a few minutes at room temperature before being introduced into the Microflex LT MALDI-TOF MS apparatus (Bruker Daltonics) for analysis. The quality of the matrix, the sample loading and the performance of the MALDI-TOF MS apparatus were controlled using the leg of an Ae. albopictus from our laboratory as a positive control.
Tissuelyser machine
Manufactured by Qiagen
Sourced in Germany
The TissueLyser is a laboratory instrument designed for efficient homogenization and disruption of biological samples, such as tissues, cells, and microorganisms, prior to nucleic acid or protein extraction. The machine uses vibration and the presence of beads or balls to break down the sample material, enabling effective sample preparation for downstream analyses.
Automatically generated - may contain errors
Lab products found in correlation
Alpha cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Maldi tof ms steel plate, by Bruker (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Cocktail tablet, by Roche (1 mentions)
Qiaamp cador pathogen mini kit, by Qiagen (1 mentions)
Atl buffer, by Qiagen (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Nd 1000 spectrophotometer, by WITec (1 mentions)
Genechip primeview human gene expression array, by Thermo Fisher Scientific (1 mentions)
3 ivt express kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
7900ht machine, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
8 protocols using tissuelyser machine
1
MALDI-TOF MS Identification of Mosquitoes
2
Quantifying Tumor IFN Levels in Mice
3
Multiplex Detection of Avian Pathogens
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About 1 g of spleen and bursa tissue samples was homogenized using a TissueLyser machine (Qiagen, Hilden, Germany) using silica beads. Upper and lower respiratory swab samples were vortexed thoroughly for 2 min. Specimen nucleic acids were extracted using the QIAamp cador Pathogen Mini Kit (Qiagen, Hilden, Germany). Specimen nucleic acids were screened for bacterial pathogens: A. paragallinarum, P. multocida, M. gallisepticum, and ORT by conventional PCR and viral pathogens (viruses causing ND, IBD, IB, and HPAI) by RT-PCR. Highly pathogenic avian influenza–positive samples were investigated for their hemagglutinin and neuraminidase identity (H5, N1, and N6 subtypes) by RT-PCR. The detection primers used in this study are listed in Supplemental Table 1 . The PCR products of the expected length were then subjected to electrophoresis in 1% agarose gel.
4
Tick DNA Extraction Protocol
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Ticks were 70%-ethanol-disinfected and then adult ticks were cut with scissors to assist DNA extraction. Specimens of H. lusitanicum were individually processed and placed into a well of a 96-well collection plate (QIAGEN, Hilden, Germany). Each vial included 3 stainless steel beads (3 mm) (Werfen, Barcelona, Spain) and 200 μL miliQ water. Plates were sealed and shaken in a TissueLyser machine (QIAGEN, Hilden, Germany) at 30 Hz (2 cycles of 6 min) for disruption and homogenization. After this, every sample (150 µL) were mixed with a solution containing 20 µL of proteinase K (QIAGEN, Hilden, Germany) and 30 µL of ATL buffer (QIAGEN, Hilden, Germany); then the mix was kept at 56 °C overnight stirring at 900 rpm.
Vaginal and anal swabs were also individually put into a well of the collection plate with a solution containing 20 µL of proteinase K and 400 µL of ATL buffer, then were kept at 56 °C overnight stirring at 900 rpm.
Vaginal and anal swabs were also individually put into a well of the collection plate with a solution containing 20 µL of proteinase K and 400 µL of ATL buffer, then were kept at 56 °C overnight stirring at 900 rpm.
5
Kidney Gene Expression Analysis
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Kidneys were isolated, frozen in liquid nitrogen and stored at –80°C. A Tissue-Lyser machine (Qiagen, Hilden, Germany) was used to homogenize the tissue. RNA was extracted from the lysed tissue using the guanidnium thiocyanate-phenol-chloroform extraction method (QIAzol lysis reagent, Qiagen, Hilden, Germany) and its concentration and quality were assessed by the Nano Drop (Witec Ag ND-1000 Spectrophotometer). cDNA was synthetized by using the PrimeScript RT reagent Kit (Takara Bio, Inc., Japan). Real-time PCRs were performed by Taqman® PCR (Applied Biosystems 7500, Foster City, CA, United States). Primers and probe mixtures (Mm00433833_mH for GR; Mm01251104_m1 for 11β-HSD2; Mn02342887_mH for Ren-1c; 4352341E for β-actin) and the Taqman Gene Expression Master Mix were purchased and used according to the manufacturer’s instructions (Applied Biosystem, Foster City, CA, United States). Each measurement was performed in duplicate. Quantification of fluorescence was performed by calculating the ΔΔCT values.
6
Transcriptome Profiling of Liver Biopsies
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Transcriptome profiling was performed with Affymetrix arrays containing 49,395 transcripts. Total RNA was extracted from snap-frozen liver biopsies obtained at baseline (n = 30) and after 4 weeks (n = 24) using the TissueLyser machine (Qiagen) and the AllPrepDNA/RNA Mini Kit (Qiagen, Hombrechtikon, Switzerland). RNA quality was assessed by capillary electrophoresis on the Agilent 2100 Bioanalyzer (Agilent Technologies, Basel, Switzerland); 100 ng was amplified and labeled using the 3’ IVT Express kit (Affymetrix). Hybridization on GeneChip PrimeView Human Gene Expression arrays (Affymetrix) was carried out according to the manufacturer’s instructions.
The data were robust multi-array normalized [15 (link)]. Pathway analysis of the genes, which were identified as differentially expressed by the microarray experiment, was undertaken using the MetaCore software (http://www.genego.com ). The gene expression data can be found in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/ ) accession no. E-MTAB-2664.
The data were robust multi-array normalized [15 (link)]. Pathway analysis of the genes, which were identified as differentially expressed by the microarray experiment, was undertaken using the MetaCore software (
7
Quantitative Gene Expression Analysis
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The mRNA was extracted from cells isolated by FACS using the Dynabead mRNA Direct kit (Invitrogen). Whole aorta tissue was homogenized using a tissue-lyser machine (Qiagen). Briefly, samples were frozen with liquid nitrogen and were homogenized with the tissue-lysser for 2 min and frozen again in liquid nitrogen. This procedure was repeated four times. Whole aorta RNA was extracted using Tri Reagent (Sigma-Aldrich), followed by RNA purification with the RNeasy mini kit (Qiagen). Reverse transcription of 500 ng RNA was performed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). QPCR experiments were conducted using SYBR Green (Applied Biosystems). Samples were loaded on a 384-well plate and analyzed on a 7900HT machine (Applied Biosystems). The expression level of each gene was determined using the relative standard curve method. The standard curve was performed using serial dilutions of mouse reference total RNA (Clontech). The expression level of each gene was calculated by interpolation from the standard curve. All values were normalized with Gapdh and Hprt1 as endogenous housekeeping genes. The sequences of the primer pairs used are listed in the Supplementary Table 1 .
8
Molecular Characterization of Plant Leaves
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Samples of the youngest leaves of 3-week old seedlings were collected at every generation. Leaves were cut into small pieces before rinsing in 70% ethanol to avoid potential contamination. A Tissue Lyser machine (Qiagen, Germany) was used to grind 2 g leaf samples with 5 mL extraction buffer. DNA precipitation was carried out using 70% ethanol and the dried pellet was re-suspended in 1X TE buffer. DNA was quantified using Nano-Drop spectrophotometry (Thermo Scientific, UK) and the DNA quality was further confirmed using 1.5% agarose gel electrophoresis at 90 V for 30 min. The polymerase chain reaction (PCR) analysis was carried out following Ashkani et al. (2013) (link) with some modifications. The PCR program used included 30 cycles of 1 min denaturation at 94°C, 1 min at 55°C annealing, 2 min polymerization at 72°C, and fast cooling to 4°C. The PCR products were identified on 3.0% agarose in 1X TBE at 80 V for 80 min and were then visualized using Molecular Imager ® (GelDocTM XR, Bio-Rad, USA).
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