The nCounter Low RNA Input Amplification Kit (NanoString Technologies, Seattle, WA) was used to retrotranscribe and pre-amplify 4 μL EV-derived RNA using 10 cycles. Retrotranscription was carried out in 0.5 mL tubes while pre-amplification, using primers targeting the genes of the Human Immunology V2 Panel (NanoString Technologies), was performed in 384-well plates to prevent sample evaporation. In parallel, a Moloney Murine Leukema Virus (M-MLV) Reverse Transcriptase Enzyme (Thermo Fisher Scientific) was also tested for cDNA synthesis. The Human Immunology V2 Panel (NanoString Technologies) was used to analyze EV-derived, pre-amplified cDNA according to manufacturer instructions. This panel targets 594 general genes involved in the immune response such as cytokines, enzymes, interferons and their receptors. Samples were hybridized for 18 h at 65 °C.
Ncounter low rna input amplification kit
Manufactured by NanoString
Sourced in United States
The NCounter Low RNA Input Amplification Kit is a laboratory equipment product designed to amplify low-input RNA samples for gene expression analysis. The kit provides a streamlined workflow to process small amounts of input RNA, enabling the detection and quantification of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Human immunology v2 panel, by NanoString (1 mentions)
M mlv reverse transcriptase enzyme, by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
Ncounter platform, by NanoString (1 mentions)
Direct zoltm rna microprep, by Zymo Research (1 mentions)
Ncounter sprint profiler, by NanoString (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Ncounter flex system, by NanoString (1 mentions)
Nsolver analysis software, by NanoString (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
6 protocols using ncounter low rna input amplification kit
1
Nanostring-Based Analysis of EV-Derived Transcripts
2
CTC RNA Extraction and Amplification Protocol
3
Purification and RNA Extraction for Transcriptome Analysis
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After purified by the TR-NanoVelcro CTC purification system, the purified cells were lysed, and the RNA was extracted using the Direct-zolTM RNA MicroPrep (Zymo Research, CA, USA) kit following the manufacturer's protocol. In brief, cells were lysed with 600 μL of Trizol solution and mixed with 600 μL of ethanol. The mixed solution was put in to Zymo-SpinTM IIC Column and centrifuged. After centrifugation the solution was washed twice with supplied wash buffer. The RNA was eluted from the column using 12 μL of RNA grade water. The amount of the eluted RNA needs to be above 2 ng/μL to pass the internal quality control. The RNA was then subjected to reverse transcription to convert to cDNA and whole transcriptome amplification using the nCounter Low RNA Input Amplification Kit (NanoString Technologies, Inc., WA, USA) following manufacturer's protocol.
4
Isolation and Analysis of CAR-T Cells
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Human CD8+ T cells were FACS-sorted from the culture with CLL cells and the enriched T-cell condition using the SH800 Cell Sorter (Sony), the cells were pelleted and total RNA was isolated using the RNeasy mini kit (Qiagen) following the manufacturers protocol. RNA was prepared with the nCounter Low RNA input amplification kit (NanoString), samples were analyzed on the nCounter SPRINT profiler using the CAR-T panel (NanoString). For further details see supplemental materials and methods.
5
Profiling Extracellular Vesicle CircRNAs
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The nCounter Low RNA Input Amplification Kit (NanoString Technologies, Seattle, WA, USA) was used to retrotranscribe and pre-amplify 4 μL of EV-derived RNA in a Verity thermal cycler (Applied Biosystems) following NanoString’s guidelines. Briefly, samples were denatured at 95 °C for 10 min and hybridized for 18 h at 67 °C. Our custom-made nCounter panel (including 78 circRNAs, 6 linear reference genes and 4 mRNAs [30 (link)] was used to analyze EV-derived pre-amplified cDNA according to the manufacturer’s instructions. RCC files containing data outputted by the NanoString nCounter Flex System (NanoString Technologies) from each run were exported to the nSolver Analysis Software (Version 4.0.70, NanoString Technologies).
6
NanoString cDNA Conversion Protocol
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cDNA conversion was performed according to the manufacturer’s protocol of the nCounter Low RNA Input Amplification kit (NanoString Technologies, LOW-RNA-48, Seattle, WA, USA). In brief, the reverse transcription (RT) master mix was prepared by combining the RT Enzyme Mix and RT Primer Mix provided. For each sample, 1 μL of RT master mix was added to 4 μL of the extracted RNA sample. The cDNA conversion program was run on an Applied Biosystems 7500 Real-Time PCR System (Life Technologies) according to the manufacturer’s protocol. The concentrations of cDNA measured in this study are shown in Table 3 .
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