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 or 608-265-9168.

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 (50-100ng/sample) and DNA (300ng/sample) 

-Amplified RNA from a small amount of sample 

-Total RNA and DNA extracted from FFPE samples 

-Crude extracts from FFPE samples 

-Cell free RNA 

-Plasma, Serum, and other Biofluids 

-Exosomes 

-LCM and Single Cell 

-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. These are the following options:

-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.

-Submit already hybridized samples: We will purify and count for you. You must drop off to TRIP all samples, kits, USB flash drive included in your kit and reagents necessary to complete your project.

-Bring your own hybridized samples and you will purify and count on the nCounter system. This option needs prior training in order to use the nCounter equipment. You must bring with you all samples, kits, USB flash drive included in your kit and reagents necessary to complete your project.

How should I prepare my samples?

How should I QC RNA samples from FFPE blocks for nCounter experiments?

Please refer to Preparing nucleic acid from FFPE samples (MAN-10050-03)

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. Below is a reference table for FFPE DV200 and corresponding recommended input.

If the sample is 50% or above roughly 200 bp, then more RNA can be loaded as a standard assay (150 instead of 100 ng total RNA) and get very similar raw counts. Once that fraction drops below 50%, increasing amounts of RNA need to get added to get equivalent counts to a fresh frozen sample.

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 to design experiments in multiples of 12.

If I only would like to rent the nCounter equipment, how should I batch process and run my samples?

We advise to plan ahead and reserve the appropriate run time for each equipment: nCounter Prep Station and nCounter Digital Analyzer, thru our iLab equipment calendar.

A run in the Prep station takes about 3 hours and a run in the Digital Analyzer takes about 4 hours per cartridge.

Only 1 cartridge can be processed at a time in the nCounter Prep Station and up to 6 cartridges can be added in the nCounter Digital Analyzer to collect data, however, one cartridge can be analyzed at a time. Data can be downloaded off the Digital Analyzer as cartridges are completed.

You can leave hybridized samples for up to 24 hours at 4C, or for several months at -80. For instance, if an overnight hybridization reaction with 36 samples (3 cartridges) is setup, you may clean up all three cartridges (one at a time) in a day in the nCounter Prep Station. Then cartridges can be loaded into the nCounter Digital Analyzer for scanning overnight or, after sealing the cartridges, put them at 4C protected from light (use foil) until the next day. If you don’t anticipate setting up the cartridges in the Prep Station all in one day, you can freeze the samples and then thaw them when you’re ready to load them into the Prep Station.

What are the nCounter platform specifications?

What is a Reporter Library File (RLF)?

Users need only upload Reporter Library Files (RLFs) when they first receive a new CodeSet Kit. The RLF is 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. Once an RLF has been uploaded, it will always be available until it is manually deleted from the Digital Analyzer. The RLF corresponding to a specific CodeSet must be uploaded before setting up a run using that CodeSet. Please bring this USB flash drive included in your kit when initiating a run at TRIP. Your data will be saved there as well.

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. Data contained in the CDF include:

IMPORTANT: The version of the RLF must match the version of the CodeSet used in the hybridization. Thus use the same USB flash drive included in your kit.

Users can create a new CDF on a personal computer or directly on the Digital Analyzer using the touchscreen. NanoString recommends users create CDFs on their computer if detailed information for each sample is required. In this case, a USB flash drive can be used to transfer the CDF to the imaging instrument.

Some example scenarios are described below.

-Scenario 1: Create a CDF on a personal computer and transfer the file to the Digital Analyzer. This is the most practical scenario if detailed and unique information is required for each sample within a cartridge. See Creating a CDF on a Personal Computer in the nCounter manual.

-Scenario 2: Create a CDF on the Digital Analyzer and enter data separately for each tube. This may be required if each sample uses a different CodeSet and required a different RLF, or if the user requires unique information for each sample. This is the most time intensive scenario. See Creating a CDF on the Digital Analyzer in the nCounter manual.

-Scenario 3: Create a CDF on the Digital Analyzer and enter data for multiple tubes simultaneously. See Creating a CDF on the Digital Analyzer in the nCounter manual.

-Scenario 4: Create a CDF on the Digital Analyzer, enter data for all tubes, and modify information for one tube. This scenario would be used if most, but not all, data were the same for samples in a single cartridge. See Creating a CDF on the Digital Analyzer in the nCounter manual.

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.