nCounter

The nCounter MAX system is a 2-machine system that allows molecular profiling of nucleic acids and protein extracted from a diverse array of samples. Gene and protein expression can be measured respectively from total RNA/DNA tissue samples, FFPE, plasma, serum and other biofluids, exosomes, whole cell lysates among others. After sample extraction and hybridization to nCounter barcodes according to the NanoString target kit of choice, samples are first setup in the nCounter Prep Station. The Prep Station is a multi-channel pipetting robot that cleans and processes samples to prepare them for data collection on the Digital Analyzer. Then, samples are transferred to the nCounter Digital Analyzer, a multi-channel epifluorescence scanner specifically configured for use with NanoString’s nCounter Cartridges. Data acquired in the nCounter Digital Analyzer is transferred to Nanostring’s open source nSolver software suite for QC and data analysis.

For nCounter MAX questions and to schedule a meeting to discuss your project and timeline, contact the TRIP Lab at trip@pathology.wisc.edu 

For pricing please see our TRIP Fee Schedule. To request nCounter MAX services, go to our iLab webpage.

FAQs

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What type of sample can I use for nCounter?

-Total RNA and DNA from fresh or FFPE tissue

-Cell free RNA 

-Plasma, Serum, and other Biofluids 

-Exosomes 

-Whole cell lysates 

What does the workflow of a Nanostring nCounter project look like?

How should I submit my samples?

There are many options and the cost will vary with the amount of labor that your project needs. Please note that for all human subjects research you must submit an approved IRB, IRB exemption and other regulatory documents to us.

-Submit tissues or separated blood serum/plasma samples to us and we can isolate nucleic acid for you and continue with the downstream assay: Hybridize, purify and count. You must drop off to TRIP all samples, kits, USB flash drive included in your kit and reagents necessary to complete your project.

-Submit your already extracted nucleic acid samples to us and we will perform the downstream assay: Hybridize, purify and count. You must drop off to TRIP all samples, kits, USB flash drive included in your kit and reagents necessary to complete your project.

It is highly recommended that samples go through QC prior to hybridization. Samples may be submitted to UWBC Gene Expression Center (GEC) (https://gec.biotech.wisc.edu/) for concentration, A260/280, A260/230, and DV200. See the FAQ tab “How should I QC RNA samples for nCounter Experiments” for more information.

How should I prepare my samples?

How should I QC RNA samples for nCounter experiments?

It is suggested to use a bioanalyzer in addition to a Nanodrop instrument. DV200 is the percent of RNA that is over 200bp of length. A DV200 measurement of minimum 20% is required for good RNA quality extracted from FFPE blocks that will go in the nCounter system.

 

The Biotech Center Gene Expression Center (GEC) has a bioanalyzer service and you can request services here.

How many samples fit in one nCounter kit cartridge?

Twelve samples fit in one cartridge, so we suggest designing experiments in multiples of 12.

What is a Reporter Library File (RLF)?

Reporter Library Files (RLFs) are the kit-specific software to run on the nCounter digital analyzer in order to get your data. The RLFs are in the USB flash drive included in your kit. Please bring this USB flash drive included in your kit when initiating a run at TRIP.

What is a Cartridge Definition File (CDF)?

Cartridge Definition File (CDF) defines sample-specific data to associate with the data output and defines the parameters the Digital Analyzer will use during image collection and processing.

Are there any batch to batch effects and what are the controls for nCounter experiments?

There are not really any batch effects from day to day, but there may be small batch effects from lot to lot.

NanoString includes three types of controls in all miRNA and mRNA panels:

-The first are negative controls, which are a set of 8 reporter and capture probes with no target. NanoString developed these controls through the ERCC (External RNA Controls Consortium). The negative controls represent the background noise and are used along with the positive controls to determine the limit of detection.

-The second are the positive controls, which are a set of six DNA control targets that are spiked in – along with their respective reporter and capture probes – at known concentrations ranging from 0.125 – 128 fM. These controls are used as a measurement of the limit of detection (the 0.5 fM positive control must be 2 SD above the mean count of the negative control probes), hybridization efficiency and pipetting accuracy (Pearson correlation of the positive controls must be >0.95), and normalization to help control for any sample to sample variation.

-The third type of controls are the housekeeping genes. Each panel includes a number of housekeeping genes. It is up to the investigator to determine which housekeeping genes in the list are most appropriate for their sample type (these genes should be robustly expressed with as little sample to sample variability as possible). Each sample is then normalized to the average of the geometric mean of all the housekeeping genes. miRNA samples normalize to the top 100 expressed miRNAs in a similar way. This housekeeping normalization should correct for lot to lot batch effects.

All data QC and normalization is done using the nSolver software after data acquisition.

Data Analysis

VIDEO: nCounter Gene Expression Analysis Overview (NanoString) 

VIDEOSnSolver Software Installation PC and Mac (NanoString) 

VIDEOSnSolver 4.0 3D Data Quick StartData Import and Data QC for Gene Expression AnalysisExperiment WizardBasic Visualizations and nSolver Advanced Analysis 2.0 visualizations (NanoString) 

nSolver 4.0 Analysis Software User Manual (MAN-C0019-08) & Quick Guide (MAN-10049-01)

nCounter Advanced Analysis(MAN-10030-03)

Rosalind Software for nCounter Data Analysis