For nanostring analysis 5uL of each RNA (20 ng/ul) to be analyzed was added to tubes in 12 strip tubes. 20ul Reporter code set diluted in buffer code set solution (NanoString Technologies), and 5uL of the Capture Probe Set (NanoString Technologies) was then added to each tube. The solution was inverted to mix, centrifuged and placed in a thermocycler (MJ Mini; Bio-rad) overnight at 65°C. Samples were removed from the thermocycler and processed in the nCounter PrepStation (NanoString Technologies). Once complete samples were transferred to the Digital Analyzer (NanoString Technologies). Expression of genes was normalized to housekeeping genes (Nanostring nCounter).
Capture probeset
Manufactured by NanoString
Sourced in United States
The Capture ProbeSet is a collection of custom-designed probes that can be used to capture and enrich target RNA molecules from biological samples. The probes are designed to specifically bind to and pull down the target RNA sequences, allowing for their subsequent detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Reporter codeset, by NanoString (3 mentions)
Digital analyzer, by NanoString (1 mentions)
Mj mini, by Bio-Rad (1 mentions)
Ncounter prep station, by NanoString (1 mentions)
Ncounter instrument, by NanoString (1 mentions)
Ncounter probe set, by NanoString (1 mentions)
Nsolver software, by NanoString (1 mentions)
Qiashredder kit, by Qiagen (1 mentions)
Ncounter mouse pancancer immune profiling panel, by NanoString (1 mentions)
Nsolver 4.0 analysis software, by NanoString (1 mentions)
Ncounter mouse pancancer pathways panel, by NanoString (1 mentions)
Ncounter flex system, by NanoString (1 mentions)
Ncounter master kit, by NanoString (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
5 protocols using capture probeset
1
NanoString Gene Expression Analysis
2
mRNA Expression Profiling via NanoString
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mRNA hybridization was set up using the 12-tube PCR hybridization strips, Reporter CodeSet and Capture ProbeSet provided by NanoString. According to the manufacturers guide, 8 µl of Master Mix (Mixture of Reporter CodeSet and Hybridizaion Buffer) was added to 5 µl of sample mRNA (altogether 150 ng extracted mRNA) in a tube. After adding 2 µl of Capture ProbeSet to each tube, the solution was gently mixed, briefly spinned and placed immediately in a pre-heated 65 °C thermal cycler for 24–26 h. After incubation, the samples were immediately placed into the nCounter Prep station, and then analysed in the Digital Analyser (nCounter FLEX Analysis System). Measurements were taken at high sensitivity with 555 FOV.
3
NanoString Analysis of T Cell Gene Expression
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Gene expression in sorted T conv cells and T reg cell populations was quantified using a custom nCounter probe set containing 301 probe pairs, including additional probes for positive and negative controls and housekeeping genes (NanoString Technologies; Yosef et al., 2013 (link)). In brief, total cellular RNA was isolated and quantitated as above, and 100 ng was hybridized with customized Reporter CodeSet and Capture ProbeSet (NanoString Technologies) by overnight incubation in a thermal cycler at 65°C. The next day, flow cell preparation and scanning were performed using an nCounter instrument (NanoString Technologies) according to the manufacturer’s instructions. Raw data were normalized using nSolver software (NanoString Technologies) and exported as raw transcript counts for presentation. To normalize data, average background was subtracted from raw count, and normalization was performed using the geometric mean count from four housekeeping genes (Gapdh, Hprt, Actb, and Tubb5). Heat maps were generated using GENE-E software.
4
NanoString nCounter Gene Expression Assay
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NanoString nCounter gene expression assay was performed using two specific probes (capture and reporter) for each gene of interest. In brief, 200 ng of total RNA per sample were hybridized with customized Reporter CodeSet and Capture ProbeSet according to the manufacturer's instructions (NanoString Technologies, Seattle, WA, USA), for direct labeling of mRNAs of interest with molecular barcodes without the use of reverse transcription or amplification. Then, the hybridized samples were recovered with the NanoString Prep Station and the mRNA molecules counted with the NanoString nCounter. The resulting counts were corrected by subtracting the average value of the negative control (alien probes from the CodeSet, lacking spiked transcript) from the raw counts obtained for each RNA. The corrected raw data were finally normalized using Arl6ip1, Polrmt, Ppia, and Rps20 as housekeeping genes [65 (link)].
5
Transcriptional Profiling of Sorted Tumor Cells
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Total RNA from sorted CD45− tumor cells was extracted using a QIAshredder kit (QIAGEN) and an RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s protocol. RNA quantification was performed using the DeNovix DS-11 Spectrophotometer (DeNovix, Inc). 100 ng of purified RNA was added to 3 μL of Reporter CodeSet and 2 μL Capture ProbeSet using an nCounter master kit as recommended (NanoString Technologies). Samples were processed on the NanoString nCounter Flex System per manufacturer instructions using the nCounter Mouse PanCancer Immune Profiling panel or the nCounter Mouse PanCancer Pathways panel (NanoString Technologies). Each gene set interrogates 750 cancer-related genes alongside 20 internal reference controls (full gene list and controls available on manufacturer’s website). Differentially expressed genes were identified in nSolver 4.0 Analysis Software (NanoString) as genes with a p value of less than 0.05 versus the respective baseline control. Reactome pathway analysis was performed using the NetworkAnalyst. The NanoString data have been deposited in the NCBI GEO under accession number GSE178135.
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