Genomic and Transcriptomic Analysis by Direct Digital Detection
BioXpedias laboraotories offer gene expression profiling using the NanoString nCounter panels. NanoString is a robust technology across many sample types and qualities, both tissues of poor and varying quality can be used including FFPE tissue sections. In collaboration with you and NanoString we find the most optimal NanoString nCounter panels and/or custom panel for your research project. We run the NanoString analyses and deliver quality controlled and normalized data to you.
The nCounter® Analysis System from NanoString Technologies offers single molecule detection with no amplification, and can be used for gene expression profiling, gene fusion, and CNV analysis. The system employs unique fluorescent barcodes that enable direct, digital detection of hundreds (≤800) of different target molecules in a single run.
NanoString is a robust and reproducible technology and can detect targets as small as 100 base pairs, and also miRNAs. It requires only nanoscale amounts of RNA and has a detection sensitivity down to 1 copy per cell.
Digital Quantification of DNA and RNA
Each assay consists of two probes of 50 bp which are complementary to the target: a capture probe linked to biotin for immobilization and purification, and a reporter probe containing a unique fluorescent barcode.
The technology is reliable due to the simplicity of the method. Once the target-probe complexes are hybridized, the complexes are captured on a streptavidin-coated surface using the biotin tag on the capture probe. Here they are aligned and immobilized for imaging, and excess probe is removed.
A scanner then digitally counts each fluorescent barcode hybridized to an RNA or DNA molecule, and puts together the counts for a total count of the target in the sample.
Thus, one count equal one molecule. This ensures a very low false positive rate (0.1%) compared to analogue technologies that measure fluorescent intensities.
The data from the digital reads are normalized to internal controls and a set of reference genes. This will generate highly precise relative counts that can be interpreted in terms of relative change across samples. Absolute counts are not calculated using this technology.
Gene Expression Profiling
Analysis of the transcriptome can be useful as a first line of investigation in explorative studies like drug target discovery or in search of biomarkers for a specific disease. The transcriptome changes rapidly in response to the external or internal environment of the body, and thus may be able to predict certain conditions.
The NanoString technology is enzyme-free with no cDNA conversion or amplification and requires as little as 25 ng of input material. It is compatible with many sample types including total RNA, plasma, serum, PBMC, FFPE and cell lysate.
Multiple panels ranging from 200-770 genes have been developed within different areas of research, including immunology, oncology and neurology. Most gene expression panels come in both a human and mouse version.
Gene Expression Panels Currently Available from NanoString:
For information about other panels or of the genes included in the panels, don’t hesitate to ask us. We are also happy to help choosing the right panel for your project. See also NanoStrings website for more panels.
miRNA Expression Profiling
In addition to gene expression panels, miRNA expression panels have also been developed for both human, mouse and rat miRNA profiling. BioXpedia offers sample analysis using NanoStrings miRNA Expression Panels.
To be able to detect the small ~22 nucleotide miRNAs, the technology has been enhanced with the use of additional specific oligos. Besides the capture and reporter probe, a target specific bridge oligo and miRTag are included in the miRNA assays. Thus, the miRNA length is extended with the miRTag by the use of the bridge oligo, and the miRNA:miRTag molecule can then hybridize to the two probes and be counted as usual.
The miRNA Expression Panels contain 827, 577 and 423 miRNA assays for human, mouse, and rat, respectively, and approximately 100 ng of total RNA is required for the analysis. Please see NanoStrings website for details on the miRNA Expression Panels.
Copy Number Variation and Gene Fusion Assays
NanoString has assays for Copy Number Variation (CNV) analysis and Gene Fusion Assays which are especially suited for FFPE tissue samples. BioXpedia offers sample analysis using the CNV and Gene Fusion panels from NanoString.
Copy Number Variation (CNV) is when a section of the genome is repeated, and where the number of repeats varies within a population. CNV can result in e.g. protein misfolding and inactivity giving rise to disease.
NanoString has developed the panel Human Cancer CN for detection of CNV of 87 genes commonly amplified or deleted in cancer. The input amount for the analysis is 300 ng genomic DNA.
Gene Fusion, as the name indicates, refers to the fusion of two separate genes by chromosomal rearrangement resulting in one hybrid gene. Gene fusions are often found to be drivers for cancer, and the resulting gene is referred to as an oncogene. NanoString has developed two gene fusion panels:
The RNA input for Gene Fusion assays is app. 100 ng of total RNA, but up to 300 ng if the RNA source is FFPE tissue.
To learn more about the NanoString nCounter Technology click here