Gene expression was measured on the nanoString nCounter SPRINT Profiler (NanoString Technologies) using the nCounter Human Immunology v2 gene expression Code Set (NanoString Technologies). Samples were prepared and processed according to the manufacturer’s recommendations. Briefly, 50 ng total RNA was hybridized in solution to the nCounter Human Immunology v2 gene expression Code Set for 18 h at 65°C. Hybridized samples were then loaded into the nCounter SPRINT cartridge (NanoString Technologies), which was then sealed and placed in the instrument for processing and analysis.
Ncounter sprint cartridge
Manufactured by NanoString
Sourced in United States
The NCounter SPRINT cartridge is a critical component of the NCounter SPRINT system, a robust and versatile gene expression analysis platform. The cartridge serves as the foundational element that enables the sensitive and precise quantification of targeted gene expression levels across a wide range of biological samples.
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Lab products found in correlation
Ncounter sprint profiler, by NanoString (4 mentions)
Ncounter gene expression assay, by NanoString (3 mentions)
Ncounter neuropathology panel, by NanoString (2 mentions)
Qubit 3.0 fluorometer, by Qiagen (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rnase free water, by Qiagen (2 mentions)
Ncounter human immunology v2 gene expression code set, by NanoString (1 mentions)
Bioanalyzer rna 6000 nano assay, by Agilent Technologies (1 mentions)
Isolate 2 rna mini kit, by Meridian Bioscience (1 mentions)
Simplyamp thermocycler, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Ncounter pancancer progression panel, by NanoString (1 mentions)
Ncounter low rna input kit, by NanoString (1 mentions)
Ncounter sprint, by NanoString (1 mentions)
Qubit rna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using ncounter sprint cartridge
1
Nanostring Immunology Gene Expression
2
Comprehensive Gene Expression Profiling of Toxoplasma Infection
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Gene Expression Assays. nCounter gene expression assays (NanoString Technologies) were performed for each of the following NanoString panels: Neuropathology, Neuroinflammation, and Inflammation+CustomizedPLUS covering a total of 1530 targeted genes including 10 parasite-specific genes: ROP18, GDA1/CD39, ADF, GRA12, SRS22A, TUBA1, SRS35A, SRS44, BAG1, and LDH2 (Geiss et al., 2008 (link)). T. gondii genes included in custom PLUS codeset were selected based on the percent expression in tachyzoite and bradyzoite growth stages (90% expression in one stage and 0% in other stage) using previous studies differentiating growth stages and ToxoDB (Goerner et al., 2020 (link)). Briefly, panel codeset probes were hybridized with 150ng of total RNA per brain over 18hr at 65C according to NanoString protocol. Inclusion of a customized PLUS codeset in the Inflammation panel required additional Reporter and Capture Plus codesets to be added during the hybridization reaction (NanoString User Manual C0019-08). Hybridized RNA was then diluted in molecular grade water and loaded into nCounter SPRINT cartridge (NanoString), placed into nCounter SPRINT Profiler, and quantified. RNA-conjugated probes were counted via NanoString Sprint Profiler technology.
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Gene Expression Assays. nCounter gene expression assays (NanoString Technologies) were performed for each of the following NanoString panels: Neuropathology, Neuroinflammation, and Inflammation+CustomizedPLUS covering a total of 1530 targeted genes including 10 parasite-specific genes: ROP18, GDA1/CD39, ADF, GRA12, SRS22A, TUBA1, SRS35A, SRS44, BAG1, and LDH2 (Geiss et al., 2008 (link)). T. gondii genes included in custom PLUS codeset were selected based on the percent expression in tachyzoite and bradyzoite growth stages (90% expression in one stage and 0% in other stage) using previous studies differentiating growth stages and ToxoDB (Goerner et al., 2020 (link)). Briefly, panel codeset probes were hybridized with 150ng of total RNA per brain over 18hr at 65C according to NanoString protocol. Inclusion of a customized PLUS codeset in the Inflammation panel required additional Reporter and Capture Plus codesets to be added during the hybridization reaction (NanoString User Manual C0019-08). Hybridized RNA was then diluted in molecular grade water and loaded into nCounter SPRINT cartridge (NanoString), placed into nCounter SPRINT Profiler, and quantified. RNA-conjugated probes were counted via NanoString Sprint Profiler technology.
3
Quantifying Fungal Gene Expression using NanoString
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Total RNA extracted from the different treatment conditions were analysed with the NanoString nCounter® analysis system [31 (link)], using a gene expression TagSet that targets 123 C.albicans genes—including three housekeeping genes, ACT1, LSC2 and THD3 [32 (link)]—and 60 C.elegans genes—including three housekeeping genes, rps-2, rps-4 and rps-23 [33 (link)]. The full list of genes with functions of the 183-genes can be found in Supplementary Table S1. Analyses of differential expression was performed using nCounter® with Elements™ XT Reagents according to manufacturer’s specifications. A multiplexed probe library (nCounter® elements CodeSet) was designed with two sequence-specific probes for each gene of interest. Probes were mixed with 100 ng of purified total RNA and allowed to hybridise (18 h, 67 °C). Samples were loaded on an nCounter® SPRINT™ Cartridge and processed with an nCounter® SPRINT™ Profiler (NanoString Technologies, USA) to quantify the transcripts. The nCounter raw expression data file (RCC) obtained was uploaded into the nSolver Analysis Software 4.0 for review of quality control metrics. The data were grouped between the experiments and control, and their expression ratios determined.
4
Gene Expression Profiling of Human ESC
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For gene profiling human ESC, pooled samples from differentiation batches of human ESC‐derived spheroids and human islets from two different preparations were sampled. Total RNA was extracted using Isolate II RNA Mini Kit (Bioline; BIO‐52072). Concentration of RNA was analyzed using nanodrop. The precise quantity and quality of RNA were determined using Bioanalyzer RNA 6000 Nano assay (Agilent; 5067‐1511). The RNA Integrity Number value for the RNA samples were between 9.9 and 10. For the gene profiling of 50 selected genes, nCounter GX Custom CodeSets (NanoString; 116000001) was used. For each sample, 50 ng of RNA was hybridized with Reporter and Capture ProbeSet in a volume of 15 μL. Hybridization was carried at 65°C for 18 hours followed by a ramp down to 4°C. The hybridization samples were then loaded on nCounter SPRINT Cartridge (nanoString; 100 078) and run on nCounter SPRINT profiler. Data were analyzed using nSolver. Recommended quality control was performed. Background was subtracted using spiked negative controls. Absolute count of RNA was further normalized using the POLR2A housekeeping gene and plotted as heat map using GraphPad prism.
5
NanoString Gene Expression Profiling
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nCounter gene expression assays (NanoString Technologies, Seattle, WA, USA) were performed using a custom NanoString Panel which probed for 100 genes. Custom panel codeset probes were hybridized with 500 ng of total RNA per large intestine mucosa sample for 18 h at 65 °C according to NanoString protocol. Molecular grade water was used to dilute hybridized RNA which was then loaded into nCounter SPRINT cartridge (NanoString, Seattle, WA, USA), and quantified. NanoString Sprint Profiler technology was utilized to count RNA-conjugated probes.
6
NanoString Neuropathology and Neuroinflammation Gene Expression Analysis
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The nCounter gene expression assays (NanoString Technologies, Seattle, WA) were performed using a custom-made combination of Neuropathology and Neuroinflammation NanoString panels. Briefly, panel code-set probes were hybridized with 150 ng of total RNA per specimen over 18hr at 65 °C in a SimplyAmp Thermocycler (Applied Biosystems, Waltham, MA), according to manufacturer’s protocol. Hybridized RNA was then diluted in molecular grade water and loaded into nCounter SPRINT cartridge (NanoString) in nCounter SPRINT Profiler, and then RNA-conjugated probes were counted via NanoString Sprint Profiler technology. Results from each panel were merged into one data file for comparative analysis. Reference gene normalization was performed for each sample by dividing each sample’s raw count profiles by the geometric mean of 8 reference genes in nSolver, as previously described (Danaher et al., 2017 (link)). Genes that did not show signal in any specimen were excluded from subsequent systems analysis.
7
Profiling DNA Damage Repair Gene Expression in PBMCs
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PBMCs (0.5 × 106) from n = 7 controls and n = 20 patients were pelleted at 1600 rpm for 8 min, lysed with RLT buffer (Qiagen), diluted 1:3 with deionized water, and stored at -80 °C until analysis. Cell lysates (80,000 cells) were hybridized with NanoString nCounter DNA Damage Repair PlexSet-96 probes and 0.2 mg/mL Proteinase K (Beckman Coulter) for 17.5 h at 67 °C. Libraries were applied to an nCounter SPRINT Cartridge and nCounter SPRINT Profiler (NanoString). Raw data were analyzed using nProfiler software (NanoString), DESeq2 [31 (link)] and RStudio (version 1.2.5019). Differentially expressed genes were classified as having a log2 FoldChange > 0.5 and a false discovery rate (FDR) < 0.05.
8
EV-derived mRNA Profiling via nCounter
9
NanoString nCounter Gene Expression Analysis
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Gene expression analysis was performed using the NanoString nCounter® Neuropathology panel (#XT-CSO-MNROP1-12, NanoString Technologies). Two panels were used: one loaded with RNA isolated from BDEVs (that were filtered during the preparation of EVs); and another one where all the samples bypassed the RNA isolation (“non-isolated”, NI) but some were filtered (F) during BDEVs preparation, while others were not (NF).
To run the first panel, 50 ng of previously isolated RNA measured by Qubit™ RNA High Sensitivity Assay Kit (Thermo Fisher) using the 3.0 QuBit Fluorometer, were mixed with RNAse–free water (Qiagen) up to 5 µL. Samples were hybridized for 18 h and mixed with 15 µL of RNAse-free water to be loaded on the nCounter® Sprint Cartridge (#LBL-10038-01, NanoStringTechnologies), following the instructions of the company. The analysis was run for 6 h.
For direct loading of EV lysates (i.e., no previous RNA isolation, NI), frozen EVs were resuspended in RLT lysis buffer and RNAse-free water in a ratio of 1:3 and loaded based on protein concentration measured by Micro BCA Protein Assay Kit (Thermo Scientific). 2.8 μg of proteins were loaded.
To run the first panel, 50 ng of previously isolated RNA measured by Qubit™ RNA High Sensitivity Assay Kit (Thermo Fisher) using the 3.0 QuBit Fluorometer, were mixed with RNAse–free water (Qiagen) up to 5 µL. Samples were hybridized for 18 h and mixed with 15 µL of RNAse-free water to be loaded on the nCounter® Sprint Cartridge (#LBL-10038-01, NanoStringTechnologies), following the instructions of the company. The analysis was run for 6 h.
For direct loading of EV lysates (i.e., no previous RNA isolation, NI), frozen EVs were resuspended in RLT lysis buffer and RNAse-free water in a ratio of 1:3 and loaded based on protein concentration measured by Micro BCA Protein Assay Kit (Thermo Scientific). 2.8 μg of proteins were loaded.
10
Gene Expression Analysis of BDEVs
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Gene expression analysis was performed by using the NanoString nCounter® Neuropathology panel (#XT-CSO-MNROP1-12, NanoString Technologies). Two panels were used: one loaded with RNA isolated from BDEVs (that were filtered during the preparation of EVs); and another one where all the samples bypassed the RNA isolation ("non-isolated", NI) but some were filtered during the BDEVs preparation (F) and the others were not filtered (NF) during preparation. To run the first panel, 50 ng of previously isolated RNA measured by Qubit TM RNA High Sensitivity Assay Kit (Thermo Fisher) using the 3.0 QuBit Fluorometer, were mixed with RNAse-free water (Qiagen) up to 5 µL. Samples were hybridized for 18 h and mixed with 15 µL of RNAse-free water to be loaded on the nCounter® Sprint Cartridge (#LBL-10038-01, NanoStringTechnologies), following the instructions of the company. The analysis was run for 6 h. For direct loading of EV lysates (i.e., no previous RNA isolation, NI), frozen EVs were resuspended in RLT lysis buffer and RNAse-free water in a ratio of 1:3 and loaded based on protein concentration measured by Micro BCA Protein Assay Kit (Thermo Scientific). 2,8 µg of proteins were loaded.
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