quantigene assay protocol  (Thermo Fisher)


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    Name:
    QuantiGene Singleplex Assay Kit
    Description:
    QuantiGene Singleplex Assays are hybridization based assays for the direct quantitation of RNA DNA or microRNA transcripts directly from cell lysates or tissue homogenates They utilize branched DNA technology for signal amplification and signal is detected using a standard luminometer The QuantiGene Singleplex Assay Kit contains all buffers plates and detection reagents necessary to perform the assay in 96 well plates Each capture plate in this assay kit is packaged individually in a sealed foil pouch
    Catalog Number:
    qs0008
    Price:
    None
    Applications:
    Gene Expression Analysis & Genotyping
    Category:
    Kits and Assays
    Buy from Supplier


    Structured Review

    Thermo Fisher quantigene assay protocol
    Validation of <t>QuantiGene</t> probes sets shows good linearity with high signal to noise ratio for several mRNA targets in different tissues. Various mRNAs were quantified from different tissue lysates. (A) Human Huntingtin ( HTT ) quantified from YAC128 mouse
    QuantiGene Singleplex Assays are hybridization based assays for the direct quantitation of RNA DNA or microRNA transcripts directly from cell lysates or tissue homogenates They utilize branched DNA technology for signal amplification and signal is detected using a standard luminometer The QuantiGene Singleplex Assay Kit contains all buffers plates and detection reagents necessary to perform the assay in 96 well plates Each capture plate in this assay kit is packaged individually in a sealed foil pouch
    https://www.bioz.com/result/quantigene assay protocol/product/Thermo Fisher
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    Images

    1) Product Images from "A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo"

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2015.0578

    Validation of QuantiGene probes sets shows good linearity with high signal to noise ratio for several mRNA targets in different tissues. Various mRNAs were quantified from different tissue lysates. (A) Human Huntingtin ( HTT ) quantified from YAC128 mouse
    Figure Legend Snippet: Validation of QuantiGene probes sets shows good linearity with high signal to noise ratio for several mRNA targets in different tissues. Various mRNAs were quantified from different tissue lysates. (A) Human Huntingtin ( HTT ) quantified from YAC128 mouse

    Techniques Used:

    Combination of TissueLyser II with QuantiGene ® bDNA: A high-throughput method of direct mRNA detection from tissues biopsies. Top panel: Punch biopsies from consecutively sectioned coronal brain samples are collected. Biopsies are incubated at
    Figure Legend Snippet: Combination of TissueLyser II with QuantiGene ® bDNA: A high-throughput method of direct mRNA detection from tissues biopsies. Top panel: Punch biopsies from consecutively sectioned coronal brain samples are collected. Biopsies are incubated at

    Techniques Used: High Throughput Screening Assay, Incubation

    Related Articles

    Amplification:

    Article Title: Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo
    Article Snippet: mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix, #QS0011). .. Cell lysates and diluted probe sets were added to the capture plate and amplification and luminescent detection were carried out as specified in the manufacturer's protocol.

    Article Title: HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions
    Article Snippet: QuantiGene 2.0 Assay Kit (Affymetrix QS0009) was used to quantitate BGLAP and the Housekeeping gene GUSB mRNA levels. .. Following an overnight incubation the capture plates were washed and the probes amplified following kit instructions.

    Article Title: Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo
    Article Snippet: mRNA quantification from cells and tissue punches mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix, #QS0011). .. Cell lysates and diluted probe sets were added to the capture plate and amplification and luminescent detection were carried out as specified in the manufacturer's protocol.

    Blocking Assay:

    Article Title: A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
    Article Snippet: .. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516) Deep 96-well sterile polypropylene plate (Corning, Axygen® , catalog number: 391-04-062) Tips (from 0.2 μl to 1 ml) (VWR) Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar) Fire-polished Pasteur pipet with cotton plug (made in-house) Tissue culture treated 10 cm dish (Corning, catalog number: 430167) 50 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352097) 15 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352098) 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22–282) 1 ml sterile syringe (BD, catalog number: 309659) Adhesive plate seals (VWR, catalog number: 60941-126) Pregnant wild-type mice (THE JACKSON LABORATORY) Cholesterol-conjugated hsiRNAs (designed and produced in-house) Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F) Poly-L-lysine (Sigma-Aldrich, catalog number: P4707) Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV) 200-Proof ethanol (Decon Labs, catalog number: 2701) DMEM cell culture medium (Mediatech, catalog number: 10-013-CV) Hibernate E (BrainBits , catalog number: HE) DNase I (Worthington , catalog number: 54M15168) Papain (Worthington , catalog number: 54N15251) Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM , catalog number: 15250061) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM , catalog number: QS0011) QuantiGene 2.0 Probe sets (varies by gene) Lysis mixture (Thermo Fisher Scientific, AffymetrixTM , catalog number: 13228) Proteinase K (Thermo Fisher Scientific, AffymetrixTM , catalog number: QS0103) Neurobasal medium NbActiv4 (BrainBits , catalog number: Nb4-500) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) NeuralQTM medium (Sigma-Aldrich, catalog number: N3100) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185) 5’UTP (Sigma-Aldrich , catalog number: U6625) 5’FdU (Sigma-Aldrich , catalog number: F3503) Papain/DNase solution (see Recipes) Plating medium (see Recipes) Feeding medium (see Recipes) .. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus) Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus) Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM , model: HeracellTM 150i) Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM , model: 1300 Series Class II, Type A2) Bunsen burner Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V) Water bath at 37 °C (Fisher Scientific, model: Model 2332) 4 °C fridge Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12) Set of two forceps (Fine Science Tools, catalog number: 11251-30) Dissection microscope (Motic, model: SMZ168 Series) Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP) Tissue culture phase-contrast inverted microscope (Motic, model: AE2000) Hemocytometer (0.1000-0.0025 mm2 ) (Neubauer) μPlate carrier (Beckman Coulter, model: SX4750) Autoplate washer (BioTek Instruments, model: ELx405) Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R) Allegra X-15R rotor (Beckman Coulter, model: SX4750) Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro)

    Article Title: HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions
    Article Snippet: QuantiGene 2.0 Assay Kit (Affymetrix QS0009) was used to quantitate BGLAP and the Housekeeping gene GUSB mRNA levels. .. A volume of 20 μl of BGLAP probeset (Affymetrix SA-10875-01) or 18 S (Affymetrix SA-10026) in blocking buffer was added to the provided capture plates before adding 80 μl of the cell lysate.

    Incubation:

    Article Title: Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
    Article Snippet: .. Cells were incubated for 72 hours at 37 °C and 5% CO2 . mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011). .. Cells were lysed in 250 μl diluted lysis mixture composed of 1 part lysis mixture (Affymetrix; #13228), 2 parts H2 O, and 0.167 μg/μl proteinase K (Affymetrix; #QS0103) for 30 minutes at 55 °C.

    Article Title: HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions
    Article Snippet: After an overnight incubation the medium was removed and serial dilutions of VDRMs in concentrations ranging from 10 μM to 2 nM in EMEM (ATCC 30–2003) + 5% charcoal dextran treated FBS were added. .. QuantiGene 2.0 Assay Kit (Affymetrix QS0009) was used to quantitate BGLAP and the Housekeeping gene GUSB mRNA levels.

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
    Article Snippet: The following day, the remainder of the QuantiGene assay protocol was performed (#QS0011; Affymetrix). .. The preamplifying solution (100 μL) was added to each well, and the plates were incubated at 55°C for 1 h. Following incubation, the plates were washed and treated with 100 μL of amplifying solution, and incubated at 55°C for 1 h. Following this step, plates were washed and incubated with 100 μL of labeling solution at 50°C for an additional hour.

    Article Title: Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity
    Article Snippet: Neurons were treated with siRNA-loaded exosomes (suspended in NeuralQ medium) and then incubated for 7 days at 37°C, 5% CO2 . .. Treated neurons were lysed and mRNAs were quantified using QuantiGene 2.0 assay kit (Affymetrix, QS0011; Thermo Fisher Scientific), and probes for mouse Htt (Affymetrix, SB-14150; Thermo Fisher Scientific) and mouse Hprt (Affymetrix, SB-15463; Thermo Fisher Scientific).

    Activity Assay:

    Article Title: Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity
    Article Snippet: Paragraph title: Measurement of siRNA Silencing Activity in Neurons ... Treated neurons were lysed and mRNAs were quantified using QuantiGene 2.0 assay kit (Affymetrix, QS0011; Thermo Fisher Scientific), and probes for mouse Htt (Affymetrix, SB-14150; Thermo Fisher Scientific) and mouse Hprt (Affymetrix, SB-15463; Thermo Fisher Scientific).

    Expressing:

    Article Title: Visualization of self-delivering hydrophobically modified siRNA cellular internalization
    Article Snippet: .. Gene expression analysis The QuantiGene 2.0 bDNA assay (Affymetrix, QS0011) was used to perform the PPIB sd-rxRNA dose response. .. HeLa cells, resuspended in DMEM with 6% FBS without antibiotics, were added into each well of a 96-well plate with a density of 5000 cells/50 μl per well. sd-rxRNA was diluted to 2× final concentration in serum free OptiMEM and 50 μl of compound was added to each well for a final volume of 100 μl/well and a final concentration of 3% FBS.

    Article Title: HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions
    Article Snippet: For bone gamma-carboxyglutamate protein (BGLAP ) expression analysis MG-63 cells, suspended in EMEM (ATCC 30–2003) + 5% charcoal dextran treated FBS, were seeded onto 96-well tissue culture treated plates at 25,000 cells per well. .. QuantiGene 2.0 Assay Kit (Affymetrix QS0009) was used to quantitate BGLAP and the Housekeeping gene GUSB mRNA levels.

    Modification:

    Article Title: Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
    Article Snippet: Cells were plated in Dulbecco's Modified Eagle's Medium with 6% FBS at 10,000 cells per well in 96-well tissue culture–treated plates. hsiRNA was diluted to four times the final concentration in OptiMEM, and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen; #13778150) was diluted to four times the final concentration (final = 0.3 µl/25 µl/well). .. Cells were incubated for 72 hours at 37 °C and 5% CO2 . mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011).

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes) .. Tissue culture hood Soft brush Ultracentrifuge 70 ml polycarbonate bottles (Beckman Coulter, catalog number: 355655) Fixed-angle Ti45 ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti, catalog number: 339160) Refrigerated ultracentrifuge (Beckman Coulter, model: Optima XE) Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra® X-15R) Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPette™ Plus) Fixed-angle TLA-110 rotor (Beckman Coulter, model: TLA-110, catalog number: 366735) 1.5 ml microcentrifuge adapters (Beckman Coulter, catalog number: 360951) Refrigerated benchtop ultracentrifuge (Beckman Coulter, model: Optima™ MAX-TL) Orbital thermo-shaker for 1.5 ml microtube (Grant Instruments, model: PHMT series) Plate reader spectrophotometer (Tecan Trading, model: Infinite® M1000 Pro) Stereotactic frame (KOPF INSTRUMENTS, model: Model 963) HPLC system with fluorescent detector with autosampler (Agilent Technologies, model: HPLC 1100 series) Hamilton syringe (Hamilton, model: Gastight #1002) Extracellular vesicles size distribution and concentration reader (Malvern Instruments, model: NanoSight NS300) Extracellular vesicles charge reader (Malvern Instruments, model: Zetasizer Nano NS) Glass micro-electrophoresis cuvette Dip Cell kit (Malvern Instruments, catalog number: ZEN1002) Transmission electron microscope (JEOL, model: JEM-1011) Note: This product has been discontinued.

    Hybridization:

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: Tips (from 0.2 μl to 1,000 μl) (VWR) Paper towel Serological pipettes individually wrapped (from 5 ml to 50 ml) (Olympus) 0.22-μm filter-sterilization system (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 567-0020) Tissue culture treated multilayer flask–T500 cm2 triple flask (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 132867) 50 ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 339652) Vacuum-connected Pasteur pipette 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282) Aluminum foil UV-transparent, flat-bottom 96-well plate (Corning, catalog number: 3635) 1 ml syringe (BD, catalog number: 309659) Parafilm (Bemis, Parafilm M® ) Whatman No. 1 filter paper (GE Healthcare, Whatman, catalog number: 10010155) 30 G ½ needle Electron microscopy grids (Electron Microscopy Sciences, catalog number: FCF2010-Ni) and clean forceps to manipulate the grid Grid storage box (Electron Microscopy Sciences, catalog number: 71156) Mouse (FVB/NJ) (THE JACKSON LABORATORY, catalog number: 001800) 200-proof ethanol (Decon Labs, catalog number: 2805M) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco™, catalog number: 14190250) Protease inhibitor cocktail stock solution (Sigma-Aldrich, catalog number: P8340) Cy3-labeled or biotinylated cholesterol-conjugated hsiRNAs (produced in-house) Cy3-OO-PNA strands, fully complementary to the hsiRNA guide strand (20 nucleotides long) (PNA Bio) Peptide-nucleic acid (PNA) hybridization assay (developed by Axolabs, Kulmbach, Germany) RIPA buffer (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 89900) Proteinase K (Thermo Fisher Scientific, Invitrogen™, catalog number: 25530049) Glycine (Sigma-Aldrich, catalog number: G7126) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153) Saponin (Sigma-Aldrich, catalog number: S1252) Note: This product has been discontinued. .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes)

    High Performance Liquid Chromatography:

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes) .. Tissue culture hood Soft brush Ultracentrifuge 70 ml polycarbonate bottles (Beckman Coulter, catalog number: 355655) Fixed-angle Ti45 ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti, catalog number: 339160) Refrigerated ultracentrifuge (Beckman Coulter, model: Optima XE) Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra® X-15R) Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPette™ Plus) Fixed-angle TLA-110 rotor (Beckman Coulter, model: TLA-110, catalog number: 366735) 1.5 ml microcentrifuge adapters (Beckman Coulter, catalog number: 360951) Refrigerated benchtop ultracentrifuge (Beckman Coulter, model: Optima™ MAX-TL) Orbital thermo-shaker for 1.5 ml microtube (Grant Instruments, model: PHMT series) Plate reader spectrophotometer (Tecan Trading, model: Infinite® M1000 Pro) Stereotactic frame (KOPF INSTRUMENTS, model: Model 963) HPLC system with fluorescent detector with autosampler (Agilent Technologies, model: HPLC 1100 series) Hamilton syringe (Hamilton, model: Gastight #1002) Extracellular vesicles size distribution and concentration reader (Malvern Instruments, model: NanoSight NS300) Extracellular vesicles charge reader (Malvern Instruments, model: Zetasizer Nano NS) Glass micro-electrophoresis cuvette Dip Cell kit (Malvern Instruments, catalog number: ZEN1002) Transmission electron microscope (JEOL, model: JEM-1011) Note: This product has been discontinued.

    Electron Microscopy:

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes) .. Tissue culture hood Soft brush Ultracentrifuge 70 ml polycarbonate bottles (Beckman Coulter, catalog number: 355655) Fixed-angle Ti45 ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti, catalog number: 339160) Refrigerated ultracentrifuge (Beckman Coulter, model: Optima XE) Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra® X-15R) Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPette™ Plus) Fixed-angle TLA-110 rotor (Beckman Coulter, model: TLA-110, catalog number: 366735) 1.5 ml microcentrifuge adapters (Beckman Coulter, catalog number: 360951) Refrigerated benchtop ultracentrifuge (Beckman Coulter, model: Optima™ MAX-TL) Orbital thermo-shaker for 1.5 ml microtube (Grant Instruments, model: PHMT series) Plate reader spectrophotometer (Tecan Trading, model: Infinite® M1000 Pro) Stereotactic frame (KOPF INSTRUMENTS, model: Model 963) HPLC system with fluorescent detector with autosampler (Agilent Technologies, model: HPLC 1100 series) Hamilton syringe (Hamilton, model: Gastight #1002) Extracellular vesicles size distribution and concentration reader (Malvern Instruments, model: NanoSight NS300) Extracellular vesicles charge reader (Malvern Instruments, model: Zetasizer Nano NS) Glass micro-electrophoresis cuvette Dip Cell kit (Malvern Instruments, catalog number: ZEN1002) Transmission electron microscope (JEOL, model: JEM-1011) Note: This product has been discontinued.

    Transfection:

    Article Title: Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
    Article Snippet: RNAiMAX and hsiRNA solutions were mixed 1:1, and 50 µl of the transfection mixture was added to 50 µl of cells resulting in 3% FBS final. .. Cells were incubated for 72 hours at 37 °C and 5% CO2 . mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011).

    Activation Assay:

    Article Title: HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions
    Article Snippet: Paragraph title: BGLAP and TRPV6 activation assays ... QuantiGene 2.0 Assay Kit (Affymetrix QS0009) was used to quantitate BGLAP and the Housekeeping gene GUSB mRNA levels.

    Protease Inhibitor:

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: Tips (from 0.2 μl to 1,000 μl) (VWR) Paper towel Serological pipettes individually wrapped (from 5 ml to 50 ml) (Olympus) 0.22-μm filter-sterilization system (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 567-0020) Tissue culture treated multilayer flask–T500 cm2 triple flask (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 132867) 50 ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 339652) Vacuum-connected Pasteur pipette 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282) Aluminum foil UV-transparent, flat-bottom 96-well plate (Corning, catalog number: 3635) 1 ml syringe (BD, catalog number: 309659) Parafilm (Bemis, Parafilm M® ) Whatman No. 1 filter paper (GE Healthcare, Whatman, catalog number: 10010155) 30 G ½ needle Electron microscopy grids (Electron Microscopy Sciences, catalog number: FCF2010-Ni) and clean forceps to manipulate the grid Grid storage box (Electron Microscopy Sciences, catalog number: 71156) Mouse (FVB/NJ) (THE JACKSON LABORATORY, catalog number: 001800) 200-proof ethanol (Decon Labs, catalog number: 2805M) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco™, catalog number: 14190250) Protease inhibitor cocktail stock solution (Sigma-Aldrich, catalog number: P8340) Cy3-labeled or biotinylated cholesterol-conjugated hsiRNAs (produced in-house) Cy3-OO-PNA strands, fully complementary to the hsiRNA guide strand (20 nucleotides long) (PNA Bio) Peptide-nucleic acid (PNA) hybridization assay (developed by Axolabs, Kulmbach, Germany) RIPA buffer (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 89900) Proteinase K (Thermo Fisher Scientific, Invitrogen™, catalog number: 25530049) Glycine (Sigma-Aldrich, catalog number: G7126) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153) Saponin (Sigma-Aldrich, catalog number: S1252) Note: This product has been discontinued. .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes)

    Transferring:

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: Tips (from 0.2 μl to 1,000 μl) (VWR) Paper towel Serological pipettes individually wrapped (from 5 ml to 50 ml) (Olympus) 0.22-μm filter-sterilization system (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 567-0020) Tissue culture treated multilayer flask–T500 cm2 triple flask (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 132867) 50 ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 339652) Vacuum-connected Pasteur pipette 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282) Aluminum foil UV-transparent, flat-bottom 96-well plate (Corning, catalog number: 3635) 1 ml syringe (BD, catalog number: 309659) Parafilm (Bemis, Parafilm M® ) Whatman No. 1 filter paper (GE Healthcare, Whatman, catalog number: 10010155) 30 G ½ needle Electron microscopy grids (Electron Microscopy Sciences, catalog number: FCF2010-Ni) and clean forceps to manipulate the grid Grid storage box (Electron Microscopy Sciences, catalog number: 71156) Mouse (FVB/NJ) (THE JACKSON LABORATORY, catalog number: 001800) 200-proof ethanol (Decon Labs, catalog number: 2805M) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco™, catalog number: 14190250) Protease inhibitor cocktail stock solution (Sigma-Aldrich, catalog number: P8340) Cy3-labeled or biotinylated cholesterol-conjugated hsiRNAs (produced in-house) Cy3-OO-PNA strands, fully complementary to the hsiRNA guide strand (20 nucleotides long) (PNA Bio) Peptide-nucleic acid (PNA) hybridization assay (developed by Axolabs, Kulmbach, Germany) RIPA buffer (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 89900) Proteinase K (Thermo Fisher Scientific, Invitrogen™, catalog number: 25530049) Glycine (Sigma-Aldrich, catalog number: G7126) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153) Saponin (Sigma-Aldrich, catalog number: S1252) Note: This product has been discontinued. .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes)

    Cell Culture:

    Article Title: A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
    Article Snippet: .. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516) Deep 96-well sterile polypropylene plate (Corning, Axygen® , catalog number: 391-04-062) Tips (from 0.2 μl to 1 ml) (VWR) Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar) Fire-polished Pasteur pipet with cotton plug (made in-house) Tissue culture treated 10 cm dish (Corning, catalog number: 430167) 50 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352097) 15 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352098) 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22–282) 1 ml sterile syringe (BD, catalog number: 309659) Adhesive plate seals (VWR, catalog number: 60941-126) Pregnant wild-type mice (THE JACKSON LABORATORY) Cholesterol-conjugated hsiRNAs (designed and produced in-house) Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F) Poly-L-lysine (Sigma-Aldrich, catalog number: P4707) Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV) 200-Proof ethanol (Decon Labs, catalog number: 2701) DMEM cell culture medium (Mediatech, catalog number: 10-013-CV) Hibernate E (BrainBits , catalog number: HE) DNase I (Worthington , catalog number: 54M15168) Papain (Worthington , catalog number: 54N15251) Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM , catalog number: 15250061) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM , catalog number: QS0011) QuantiGene 2.0 Probe sets (varies by gene) Lysis mixture (Thermo Fisher Scientific, AffymetrixTM , catalog number: 13228) Proteinase K (Thermo Fisher Scientific, AffymetrixTM , catalog number: QS0103) Neurobasal medium NbActiv4 (BrainBits , catalog number: Nb4-500) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) NeuralQTM medium (Sigma-Aldrich, catalog number: N3100) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185) 5’UTP (Sigma-Aldrich , catalog number: U6625) 5’FdU (Sigma-Aldrich , catalog number: F3503) Papain/DNase solution (see Recipes) Plating medium (see Recipes) Feeding medium (see Recipes) .. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus) Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus) Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM , model: HeracellTM 150i) Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM , model: 1300 Series Class II, Type A2) Bunsen burner Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V) Water bath at 37 °C (Fisher Scientific, model: Model 2332) 4 °C fridge Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12) Set of two forceps (Fine Science Tools, catalog number: 11251-30) Dissection microscope (Motic, model: SMZ168 Series) Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP) Tissue culture phase-contrast inverted microscope (Motic, model: AE2000) Hemocytometer (0.1000-0.0025 mm2 ) (Neubauer) μPlate carrier (Beckman Coulter, model: SX4750) Autoplate washer (BioTek Instruments, model: ELx405) Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R) Allegra X-15R rotor (Beckman Coulter, model: SX4750) Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro)

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes) .. Tissue culture hood Soft brush Ultracentrifuge 70 ml polycarbonate bottles (Beckman Coulter, catalog number: 355655) Fixed-angle Ti45 ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti, catalog number: 339160) Refrigerated ultracentrifuge (Beckman Coulter, model: Optima XE) Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra® X-15R) Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPette™ Plus) Fixed-angle TLA-110 rotor (Beckman Coulter, model: TLA-110, catalog number: 366735) 1.5 ml microcentrifuge adapters (Beckman Coulter, catalog number: 360951) Refrigerated benchtop ultracentrifuge (Beckman Coulter, model: Optima™ MAX-TL) Orbital thermo-shaker for 1.5 ml microtube (Grant Instruments, model: PHMT series) Plate reader spectrophotometer (Tecan Trading, model: Infinite® M1000 Pro) Stereotactic frame (KOPF INSTRUMENTS, model: Model 963) HPLC system with fluorescent detector with autosampler (Agilent Technologies, model: HPLC 1100 series) Hamilton syringe (Hamilton, model: Gastight #1002) Extracellular vesicles size distribution and concentration reader (Malvern Instruments, model: NanoSight NS300) Extracellular vesicles charge reader (Malvern Instruments, model: Zetasizer Nano NS) Glass micro-electrophoresis cuvette Dip Cell kit (Malvern Instruments, catalog number: ZEN1002) Transmission electron microscope (JEOL, model: JEM-1011) Note: This product has been discontinued.

    other:

    Article Title: Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs
    Article Snippet: LOPAC1280 small molecule secondary screen The QuantiGene 2.0 bDNA assay (Affymetrix, QS0011) was used to perform a secondary validation screen on the primary hits.

    Sequencing:

    Article Title: mRNA treatment produces sustained expression of enzymatically active human ADAMTS13 in mice
    Article Snippet: Plasma and liver mRNA quantification mRNA from mouse plasma or liver tissue lysates were quantitatively measured with branched DNA (bDNA) assay using QuantiGene Singleplex kit (Affymetrix, Santa Clara, CA). .. A panel of detection probes were designed against the target mRNA sequence, assay was validated as instructed by the manufacturer using purified mRNA as reference standard prior to quantitating the mRNA within mouse plasma or liver tissue lysates.

    In Vivo:

    Article Title: Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing
    Article Snippet: For in vivo silencing of Htt mRNA, mice were treated with aCSF (100 µl per pump, n = 9 mice), 20–30 × 109 particles/day of exosomes alone (100 µl per pump, n = 9 mice), 1 µg/day of NTC siRNA associated with 20–30 × 109 particles/day of exosomes (100 µl per pump, n = 10 mice), 0.5 or 1 µg/day hsiRNAHTT alone (100 µl per pump, n = 10 mice and n = 8 mice per group, respectively), and 0.5 or 1 µg/day of hsiRNAHTT associated with 20–30 × 109 particles/day of exosomes (100 µl per pump, n = 9 mice and n = 6 mice per group, respectively). .. The mRNA levels were quantified using the QuantiGene 2.0 Assay (Affymetrix, Santa Clara, CA; #QS0011).

    Article Title: 5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo
    Article Snippet: Cells were lysed and mRNA quantification was performed using the QuantiGene 2.0 assay kit (Affymetrix, #QS0011) as described previously ( ). .. For in vivo experiments, mice were euthanized and organs placed in RNAlater (Sigma, #R0901) at 4°C overnight.

    Isolation:

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
    Article Snippet: Total RNA was isolated using the mir Vana™ miRNA isolation kit (#AM1560; Life Technologies), eluted in 100 μL of RNase free water, and diluted to a volume of 260 μL. .. The following day, the remainder of the QuantiGene assay protocol was performed (#QS0011; Affymetrix).

    Flow Cytometry:

    Article Title: Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing
    Article Snippet: The mRNA levels were quantified using the QuantiGene 2.0 Assay (Affymetrix, Santa Clara, CA; #QS0011). .. The mRNA levels were quantified using the QuantiGene 2.0 Assay (Affymetrix, Santa Clara, CA; #QS0011).

    Labeling:

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
    Article Snippet: The following day, the remainder of the QuantiGene assay protocol was performed (#QS0011; Affymetrix). .. The preamplifying solution (100 μL) was added to each well, and the plates were incubated at 55°C for 1 h. Following incubation, the plates were washed and treated with 100 μL of amplifying solution, and incubated at 55°C for 1 h. Following this step, plates were washed and incubated with 100 μL of labeling solution at 50°C for an additional hour.

    Mouse Assay:

    Article Title: A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
    Article Snippet: .. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516) Deep 96-well sterile polypropylene plate (Corning, Axygen® , catalog number: 391-04-062) Tips (from 0.2 μl to 1 ml) (VWR) Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar) Fire-polished Pasteur pipet with cotton plug (made in-house) Tissue culture treated 10 cm dish (Corning, catalog number: 430167) 50 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352097) 15 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352098) 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22–282) 1 ml sterile syringe (BD, catalog number: 309659) Adhesive plate seals (VWR, catalog number: 60941-126) Pregnant wild-type mice (THE JACKSON LABORATORY) Cholesterol-conjugated hsiRNAs (designed and produced in-house) Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F) Poly-L-lysine (Sigma-Aldrich, catalog number: P4707) Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV) 200-Proof ethanol (Decon Labs, catalog number: 2701) DMEM cell culture medium (Mediatech, catalog number: 10-013-CV) Hibernate E (BrainBits , catalog number: HE) DNase I (Worthington , catalog number: 54M15168) Papain (Worthington , catalog number: 54N15251) Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM , catalog number: 15250061) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM , catalog number: QS0011) QuantiGene 2.0 Probe sets (varies by gene) Lysis mixture (Thermo Fisher Scientific, AffymetrixTM , catalog number: 13228) Proteinase K (Thermo Fisher Scientific, AffymetrixTM , catalog number: QS0103) Neurobasal medium NbActiv4 (BrainBits , catalog number: Nb4-500) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) NeuralQTM medium (Sigma-Aldrich, catalog number: N3100) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185) 5’UTP (Sigma-Aldrich , catalog number: U6625) 5’FdU (Sigma-Aldrich , catalog number: F3503) Papain/DNase solution (see Recipes) Plating medium (see Recipes) Feeding medium (see Recipes) .. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus) Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus) Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM , model: HeracellTM 150i) Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM , model: 1300 Series Class II, Type A2) Bunsen burner Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V) Water bath at 37 °C (Fisher Scientific, model: Model 2332) 4 °C fridge Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12) Set of two forceps (Fine Science Tools, catalog number: 11251-30) Dissection microscope (Motic, model: SMZ168 Series) Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP) Tissue culture phase-contrast inverted microscope (Motic, model: AE2000) Hemocytometer (0.1000-0.0025 mm2 ) (Neubauer) μPlate carrier (Beckman Coulter, model: SX4750) Autoplate washer (BioTek Instruments, model: ELx405) Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R) Allegra X-15R rotor (Beckman Coulter, model: SX4750) Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro)

    Article Title: Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing
    Article Snippet: Mice were killed 6 hours or 7 days later. mRNA quantification. .. The mRNA levels were quantified using the QuantiGene 2.0 Assay (Affymetrix, Santa Clara, CA; #QS0011).

    Article Title: 5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo
    Article Snippet: Cells were lysed and mRNA quantification was performed using the QuantiGene 2.0 assay kit (Affymetrix, #QS0011) as described previously ( ). .. For in vivo experiments, mice were euthanized and organs placed in RNAlater (Sigma, #R0901) at 4°C overnight.

    Lysis:

    Article Title: A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
    Article Snippet: .. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516) Deep 96-well sterile polypropylene plate (Corning, Axygen® , catalog number: 391-04-062) Tips (from 0.2 μl to 1 ml) (VWR) Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar) Fire-polished Pasteur pipet with cotton plug (made in-house) Tissue culture treated 10 cm dish (Corning, catalog number: 430167) 50 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352097) 15 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352098) 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22–282) 1 ml sterile syringe (BD, catalog number: 309659) Adhesive plate seals (VWR, catalog number: 60941-126) Pregnant wild-type mice (THE JACKSON LABORATORY) Cholesterol-conjugated hsiRNAs (designed and produced in-house) Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F) Poly-L-lysine (Sigma-Aldrich, catalog number: P4707) Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV) 200-Proof ethanol (Decon Labs, catalog number: 2701) DMEM cell culture medium (Mediatech, catalog number: 10-013-CV) Hibernate E (BrainBits , catalog number: HE) DNase I (Worthington , catalog number: 54M15168) Papain (Worthington , catalog number: 54N15251) Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM , catalog number: 15250061) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM , catalog number: QS0011) QuantiGene 2.0 Probe sets (varies by gene) Lysis mixture (Thermo Fisher Scientific, AffymetrixTM , catalog number: 13228) Proteinase K (Thermo Fisher Scientific, AffymetrixTM , catalog number: QS0103) Neurobasal medium NbActiv4 (BrainBits , catalog number: Nb4-500) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) NeuralQTM medium (Sigma-Aldrich, catalog number: N3100) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185) 5’UTP (Sigma-Aldrich , catalog number: U6625) 5’FdU (Sigma-Aldrich , catalog number: F3503) Papain/DNase solution (see Recipes) Plating medium (see Recipes) Feeding medium (see Recipes) .. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus) Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus) Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM , model: HeracellTM 150i) Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM , model: 1300 Series Class II, Type A2) Bunsen burner Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V) Water bath at 37 °C (Fisher Scientific, model: Model 2332) 4 °C fridge Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12) Set of two forceps (Fine Science Tools, catalog number: 11251-30) Dissection microscope (Motic, model: SMZ168 Series) Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP) Tissue culture phase-contrast inverted microscope (Motic, model: AE2000) Hemocytometer (0.1000-0.0025 mm2 ) (Neubauer) μPlate carrier (Beckman Coulter, model: SX4750) Autoplate washer (BioTek Instruments, model: ELx405) Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R) Allegra X-15R rotor (Beckman Coulter, model: SX4750) Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro)

    Article Title: Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo
    Article Snippet: mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix, #QS0011). .. Cells were lysed in 250 μl diluted lysis mixture with proteinase K (Affymetrix, #13228) for 30 min at 55°C.

    Article Title: Visualization of self-delivering hydrophobically modified siRNA cellular internalization
    Article Snippet: Gene expression analysis The QuantiGene 2.0 bDNA assay (Affymetrix, QS0011) was used to perform the PPIB sd-rxRNA dose response. .. Briefly, cells were lysed in 250 μl diluted lysis mixture (1:2 lysis mixture:water) with 0.167 ug/μl proteinase K (Affymetrix QS0103) for 30 min at 55°C.

    Article Title: Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
    Article Snippet: Cells were incubated for 72 hours at 37 °C and 5% CO2 . mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011). .. Cells were lysed in 250 μl diluted lysis mixture composed of 1 part lysis mixture (Affymetrix; #13228), 2 parts H2 O, and 0.167 μg/μl proteinase K (Affymetrix; #QS0103) for 30 minutes at 55 °C.

    Article Title: HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions
    Article Snippet: QuantiGene 2.0 Assay Kit (Affymetrix QS0009) was used to quantitate BGLAP and the Housekeeping gene GUSB mRNA levels. .. Following the kit instructions, lysis buffer was added to the frozen cells and incubated briefly at 55 °C.

    Article Title: RNAi Modulation of Placental sFLT1 for the Treatment of Preeclampsia.
    Article Snippet: mRNA quantification in cells and tissue punches mRNA was quantified from both cells and tissue punches using the QuantiGene 2.0 assay kit (Affymetrix, # QS0011) as described previously , . .. Cells were lysed in 250 μL diluted lysis mixture composed of 1 part lysis mixture (Affymetrix, #13228), 2 parts H2 O, and 0.167 μg/μl proteinase K (Affymetrix,# QS0103) for 30 minutes at 55°C.

    Article Title: Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo
    Article Snippet: mRNA quantification from cells and tissue punches mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix, #QS0011). .. Cells were lysed in 250 μl diluted lysis mixture with proteinase K (Affymetrix, #13228) for 30 min at 55°C.

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
    Article Snippet: The pooled tissue punches were homogenized by electronic pestle in 300 μL of lysis buffer. .. The following day, the remainder of the QuantiGene assay protocol was performed (#QS0011; Affymetrix).

    Article Title: Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing
    Article Snippet: The mRNA levels were quantified using the QuantiGene 2.0 Assay (Affymetrix, Santa Clara, CA; #QS0011). .. For in vitro experiments, cells were plated at 1 × 105 neurons per well in 96-well plate and lysed in 250 μl diluted lysis mixture per well (Affymetrix; #13228) supplemented with 0.167 μg/μl proteinase K (Affymetrix; #QS0103) for 30 minutes at 55 °C.

    Purification:

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
    Article Snippet: For extraction of total purified RNA, three striatum punches and three cortical punches from the left side of the brain were pooled into separate tubes. .. The following day, the remainder of the QuantiGene assay protocol was performed (#QS0011; Affymetrix).

    Article Title: mRNA treatment produces sustained expression of enzymatically active human ADAMTS13 in mice
    Article Snippet: Plasma and liver mRNA quantification mRNA from mouse plasma or liver tissue lysates were quantitatively measured with branched DNA (bDNA) assay using QuantiGene Singleplex kit (Affymetrix, Santa Clara, CA). .. A panel of detection probes were designed against the target mRNA sequence, assay was validated as instructed by the manufacturer using purified mRNA as reference standard prior to quantitating the mRNA within mouse plasma or liver tissue lysates.

    Sample Prep:

    Article Title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo
    Article Snippet: Paragraph title: Sample preparation ... The following day, the remainder of the QuantiGene assay protocol was performed (#QS0011; Affymetrix).

    In Vitro:

    Article Title: Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
    Article Snippet: The primary screen for active Htt siRNAs, therefore, was performed at 1.5 µmol/l compound, which also served as the maximal dose for in vitro dose–response assays. hsiRNA lipid-mediated delivery. .. Cells were incubated for 72 hours at 37 °C and 5% CO2 . mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011).

    Article Title: Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing
    Article Snippet: The mRNA levels were quantified using the QuantiGene 2.0 Assay (Affymetrix, Santa Clara, CA; #QS0011). .. For in vitro experiments, cells were plated at 1 × 105 neurons per well in 96-well plate and lysed in 250 μl diluted lysis mixture per well (Affymetrix; #13228) supplemented with 0.167 μg/μl proteinase K (Affymetrix; #QS0103) for 30 minutes at 55 °C.

    Article Title: 5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo
    Article Snippet: Tissue punches were lysed and mRNA quantification was performed using the QuantiGene 2.0 assay kit (Affymetrix, #QS0011) as described previously ( ). .. In both in vitro and in vivo experiments, hsiRNAPPIB was used as non-targeting control (NTC) for HTT silencing, and hsiRNAHTT was used as non-targeting control (NTC) for PPIB silencing.

    Produced:

    Article Title: A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
    Article Snippet: .. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516) Deep 96-well sterile polypropylene plate (Corning, Axygen® , catalog number: 391-04-062) Tips (from 0.2 μl to 1 ml) (VWR) Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar) Fire-polished Pasteur pipet with cotton plug (made in-house) Tissue culture treated 10 cm dish (Corning, catalog number: 430167) 50 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352097) 15 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352098) 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22–282) 1 ml sterile syringe (BD, catalog number: 309659) Adhesive plate seals (VWR, catalog number: 60941-126) Pregnant wild-type mice (THE JACKSON LABORATORY) Cholesterol-conjugated hsiRNAs (designed and produced in-house) Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F) Poly-L-lysine (Sigma-Aldrich, catalog number: P4707) Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV) 200-Proof ethanol (Decon Labs, catalog number: 2701) DMEM cell culture medium (Mediatech, catalog number: 10-013-CV) Hibernate E (BrainBits , catalog number: HE) DNase I (Worthington , catalog number: 54M15168) Papain (Worthington , catalog number: 54N15251) Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM , catalog number: 15250061) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM , catalog number: QS0011) QuantiGene 2.0 Probe sets (varies by gene) Lysis mixture (Thermo Fisher Scientific, AffymetrixTM , catalog number: 13228) Proteinase K (Thermo Fisher Scientific, AffymetrixTM , catalog number: QS0103) Neurobasal medium NbActiv4 (BrainBits , catalog number: Nb4-500) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) NeuralQTM medium (Sigma-Aldrich, catalog number: N3100) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185) 5’UTP (Sigma-Aldrich , catalog number: U6625) 5’FdU (Sigma-Aldrich , catalog number: F3503) Papain/DNase solution (see Recipes) Plating medium (see Recipes) Feeding medium (see Recipes) .. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus) Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus) Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM , model: HeracellTM 150i) Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM , model: 1300 Series Class II, Type A2) Bunsen burner Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V) Water bath at 37 °C (Fisher Scientific, model: Model 2332) 4 °C fridge Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12) Set of two forceps (Fine Science Tools, catalog number: 11251-30) Dissection microscope (Motic, model: SMZ168 Series) Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP) Tissue culture phase-contrast inverted microscope (Motic, model: AE2000) Hemocytometer (0.1000-0.0025 mm2 ) (Neubauer) μPlate carrier (Beckman Coulter, model: SX4750) Autoplate washer (BioTek Instruments, model: ELx405) Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R) Allegra X-15R rotor (Beckman Coulter, model: SX4750) Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro)

    Article Title: Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Article Snippet: Tips (from 0.2 μl to 1,000 μl) (VWR) Paper towel Serological pipettes individually wrapped (from 5 ml to 50 ml) (Olympus) 0.22-μm filter-sterilization system (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 567-0020) Tissue culture treated multilayer flask–T500 cm2 triple flask (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 132867) 50 ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 339652) Vacuum-connected Pasteur pipette 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282) Aluminum foil UV-transparent, flat-bottom 96-well plate (Corning, catalog number: 3635) 1 ml syringe (BD, catalog number: 309659) Parafilm (Bemis, Parafilm M® ) Whatman No. 1 filter paper (GE Healthcare, Whatman, catalog number: 10010155) 30 G ½ needle Electron microscopy grids (Electron Microscopy Sciences, catalog number: FCF2010-Ni) and clean forceps to manipulate the grid Grid storage box (Electron Microscopy Sciences, catalog number: 71156) Mouse (FVB/NJ) (THE JACKSON LABORATORY, catalog number: 001800) 200-proof ethanol (Decon Labs, catalog number: 2805M) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco™, catalog number: 14190250) Protease inhibitor cocktail stock solution (Sigma-Aldrich, catalog number: P8340) Cy3-labeled or biotinylated cholesterol-conjugated hsiRNAs (produced in-house) Cy3-OO-PNA strands, fully complementary to the hsiRNA guide strand (20 nucleotides long) (PNA Bio) Peptide-nucleic acid (PNA) hybridization assay (developed by Axolabs, Kulmbach, Germany) RIPA buffer (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 89900) Proteinase K (Thermo Fisher Scientific, Invitrogen™, catalog number: 25530049) Glycine (Sigma-Aldrich, catalog number: G7126) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153) Saponin (Sigma-Aldrich, catalog number: S1252) Note: This product has been discontinued. .. Glutaraldehyde (Polysciences, catalog number: 01909-10) Avertin (Sigma-Aldrich, catalog number: T48402) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, Invitrogen™, catalog number: QS0011) Quantigene 2.0 Probesets (Varies by gene) Dulbecco’s modified Eagle medium (DMEM) MSC culture media (ATCC, catalog number: PCS-500-041) Sucrose (Sigma-Aldrich, catalog number: S0389) Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758) Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758) Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) Sodium phosphate monobasic monohydrate (NaH2 PO4 .H2 O) Sodium phosphate monobasic (NaH2 PO4 ) (Sigma-Aldrich, catalog number: S3139) Acetonitrile (50% acetonitrile solution, diluted in distilled H2 O) (Fisher Scientific, catalog number: A998-4) Sodium perchlorate monohydrate (NaClO4 .xH2 O) (Fisher Scientific, catalog number: S490-500) Methyl cellulose (Sigma-Aldrich, catalog number: M6385) PFA powder (Sigma-Aldrich, catalog number: P6148) Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400) Oxalic acid Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153) Ammonium hydroxide (NH4 OH) (Fisher Scientific, catalog number: A669-212) Cell culture medium (see Recipes) 1 M sucrose (see Recipes) 1 M Tris-HCl (see Recipes) 0.5 M EDTA (see Recipes) 3 M KCl (see Recipes) 10% SDS (see Recipes) 0.1 M sodium phosphate buffer (see Recipes) HPLC buffer A (see Recipes) HPLC buffer B (see Recipes) Glutaraldehyde, 1% (v/v) (see Recipes) Methyl cellulose, 2% (w/v) (see Recipes) Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes) Uranyl acetate (4% w/v), pH 4 (see Recipes) Uranyl-oxalate, pH 7 (see Recipes) Methyl cellulose-UA, pH 4 (see Recipes)

    Concentration Assay:

    Article Title: Visualization of self-delivering hydrophobically modified siRNA cellular internalization
    Article Snippet: Gene expression analysis The QuantiGene 2.0 bDNA assay (Affymetrix, QS0011) was used to perform the PPIB sd-rxRNA dose response. .. HeLa cells, resuspended in DMEM with 6% FBS without antibiotics, were added into each well of a 96-well plate with a density of 5000 cells/50 μl per well. sd-rxRNA was diluted to 2× final concentration in serum free OptiMEM and 50 μl of compound was added to each well for a final volume of 100 μl/well and a final concentration of 3% FBS.

    Article Title: Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain
    Article Snippet: Cells were plated in Dulbecco's Modified Eagle's Medium with 6% FBS at 10,000 cells per well in 96-well tissue culture–treated plates. hsiRNA was diluted to four times the final concentration in OptiMEM, and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen; #13778150) was diluted to four times the final concentration (final = 0.3 µl/25 µl/well). .. Cells were incubated for 72 hours at 37 °C and 5% CO2 . mRNA quantification in cells and tissue punches. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix; #QS0011).

    Staining:

    Article Title: A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
    Article Snippet: .. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516) Deep 96-well sterile polypropylene plate (Corning, Axygen® , catalog number: 391-04-062) Tips (from 0.2 μl to 1 ml) (VWR) Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar) Fire-polished Pasteur pipet with cotton plug (made in-house) Tissue culture treated 10 cm dish (Corning, catalog number: 430167) 50 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352097) 15 ml conical centrifuge tubes (Corning, Falcon® , catalog number: 352098) 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22–282) 1 ml sterile syringe (BD, catalog number: 309659) Adhesive plate seals (VWR, catalog number: 60941-126) Pregnant wild-type mice (THE JACKSON LABORATORY) Cholesterol-conjugated hsiRNAs (designed and produced in-house) Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F) Poly-L-lysine (Sigma-Aldrich, catalog number: P4707) Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV) 200-Proof ethanol (Decon Labs, catalog number: 2701) DMEM cell culture medium (Mediatech, catalog number: 10-013-CV) Hibernate E (BrainBits , catalog number: HE) DNase I (Worthington , catalog number: 54M15168) Papain (Worthington , catalog number: 54N15251) Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM , catalog number: 15250061) QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM , catalog number: QS0011) QuantiGene 2.0 Probe sets (varies by gene) Lysis mixture (Thermo Fisher Scientific, AffymetrixTM , catalog number: 13228) Proteinase K (Thermo Fisher Scientific, AffymetrixTM , catalog number: QS0103) Neurobasal medium NbActiv4 (BrainBits , catalog number: Nb4-500) Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV) NeuralQTM medium (Sigma-Aldrich, catalog number: N3100) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185) 5’UTP (Sigma-Aldrich , catalog number: U6625) 5’FdU (Sigma-Aldrich , catalog number: F3503) Papain/DNase solution (see Recipes) Plating medium (see Recipes) Feeding medium (see Recipes) .. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus) Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus) Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM , model: HeracellTM 150i) Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM , model: 1300 Series Class II, Type A2) Bunsen burner Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V) Water bath at 37 °C (Fisher Scientific, model: Model 2332) 4 °C fridge Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12) Set of two forceps (Fine Science Tools, catalog number: 11251-30) Dissection microscope (Motic, model: SMZ168 Series) Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP) Tissue culture phase-contrast inverted microscope (Motic, model: AE2000) Hemocytometer (0.1000-0.0025 mm2 ) (Neubauer) μPlate carrier (Beckman Coulter, model: SX4750) Autoplate washer (BioTek Instruments, model: ELx405) Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R) Allegra X-15R rotor (Beckman Coulter, model: SX4750) Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro)

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    Thermo Fisher viewrna mirna ish cell assay kit
    Viewrna Mirna Ish Cell Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/viewrna mirna ish cell assay kit/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
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    viewrna mirna ish cell assay kit - by Bioz Stars, 2020-04
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