Resources

Nanostring nCounter SPRINT platform

We are the only Spanish collaborating center of Nanostring!

Platform for biomarkers discovery and validation in the field of transcriptomics and proteomics (and CNVs for DNA). Nanostring nCounter® analysis system can directly analyse hundreds of mRNAs, miRNAs, CNVs or proteins through a molecular digital measurement in a single tube and in absence of any enzymatic reaction (no amplification, no library preparation or retro-transcription is needed). This is a high sensibility and reproducibility technology with a high multiplexing capability (up to 800 genes in a single reaction), which not only reduces the number of reactions but also saves on the required sample input amount. The nCounter Analysis System utilizes a novel digital count through a colour barcode technology for direct multiplexed measurement. The technology uses molecular "barcodes" and single molecule imaging for the direct hybridization and detection of hundreds of unique transcripts in a single reaction. Each color-coded barcode is attached to a single target-specific probe corresponding to a molecule of interest. You will obtain the real number of target molecules presents in a given sample.

Nanostring technology has a great number of potential applications in different areas of the biomedical research field. There are many pre-build panels (12 samples) available in different research fields like oncology, immunology, neurology, etc… You also have the chance of ordering custom panels with your targets of interest (up to 800 targets).

Nanostring leads simultaneous quantification of proteins and gene expression in the same sample with nCounter® Vantage 3D™ Immune Assays. You can study mRNA and proteins of the cellular surface like receptors, cellular lineage markers or regulatory molecules to investigate changes in cellular type abundances and activation status. In addition, it offers you the opportunity to study mRNA and some intracellular protein levels like cytokines, chemokines and transcription factors involved in initial stages of cellular communication and immune differentiation.

From our laboratory, and with our experts, we offer you support and advice with the design of the experiment you need to carry out and the development of the protocol. In addition, we perform all the experimental part of your research project,. Finally, we can also offer support in the analysis and interpretation of results, delivering results in a simple format and in an understandable language. Our scientific and bioinformatics experts analyze, and interpret the results of complex “omic” studies, so that we can approach a comprehensive analysis of the raw data to make it easily interpretable. We have developed our own analysis pipelines in R to offer the best solutions to the scientific community.

Nanostring nCounter service

• Support with experimental panel design and protocol development.
• Reagents, Codesets and cartridges ordering.
• RNA/DNA quality/quantity assessment.
• Preparation and processing of samples, hybridization and imaging.
• Results in csv file format containing the raw and normalized data.
• Support with the data analysis: differential expression analysis; over-representation (ORA) and gene-set enrichment (GSEA) analysis using R software.

The IDIS Proteomics unit was created with the aim to enhancing, supporting and offering researchers from both IDIS and other public and private organizations, a complete infrastructure in the field of proteomics. This platform is equipped with the most innovative technology that enables studies to be carried out on the characterization of global proteomes, as well as differential expression analysis studies. Bearing in mind that the cause of most human diseases is due to the functional deregulation of the proteins interaction, the most important clinical application would be the identification of biomarkers diseases.

Equipment

• White room, to avoid Keratin contamination during the simple preparation.
• Two-dimensional gel electrophoresis
  • • “Protean IEF system (BioRad). Protein separation by their Isoelectric point
    • “Mini Protean 3 electrophoresis system” (BioRad), “Protean IIXL Cell” (BioRad), y “Protean Plus Dodeca Cell” (BioRad, It is posible run 12 simultaneus gels). Protein separation by their molecular weight
• DIGE Imager (GE Healthcare) for scanning gels stained with fluorescent DIGE or SYPRO, etc.
• Analysis software sameSpots™ dimensional gels (Nonlinear). This program allows the identification, quantification and analysis of the protein spots present in the 2-DE gels.
• Mass spectrometer MALDI-TOF/TOF (4800 Analyzer, Applied Biosystems). The MALDI-TOF / TOF system allows the identification of proteins by means of the analysis of the masses of the peptides generated by enzymatic digestion of proteins (PMF), and the fragmentation of the peptides. It also allows the determination with great sensitivity and accuracy of the molecular weight of proteins, peptides, oligonucleotides and other biomolecules, as well as small molecules.
• Nano-HPLC (nanoLC Ultra 1D plus, ABSciex) coupled to a spoter eksigent (ABSciex)

Allows preparation for MALDI analysis of separated peptide fractions by liquid nanochromatography, automating proteomic workflow, from chromatographic separation to identification and characterization by mass spectrometry using the MALDI TOF / TOF system

• Triple TOF 6600 (ABSciex) coupled to a nanoLC425 (Eksigent)

It allows the separation of complex samples of proteins for the identification and characterization of proteins by LC-MS / MS technology

• GPS, Global Proteomics Server, specialized software with search engine (Mascot) to perform the protein identifications.

This software has an integrated DeNovo Explorer software that allows de novo sequencing from MS / MS fragmentation data

• Software “ProteinPilot” (AbSciex) that allows the quantification of labeled peptides (ICAT, SILAC) in quantitative proteomics studies. this software is also used in the identification of proteins using the paragon algorithm.
• Software “Markerview 1.7” (AbSciex) which allows the analysis of main components between samples (PCA) and analysis of SWATH assays
• Analyst 1.7 (AbSciex) acquisition software for the triple TOF 6600 that allows the design of the programs of DDA, SWATH, SRM
• Software “peakview” (AbSciex) which allows the analysis and revision of the LC-MS and the MSMS of the samples acquired with the Triple TOF, as well as the SWATH analysis.
• PEAKS 8.5 (Peaks) allows Novo sequencing from the MSMS and search for all types of post-translational modifications

MALDI-TOF/TOF machine for Molecular Mass determination (DMM)

Proteomics using peptide mass fingerprint (PMF)

Proteomics using 6600 Triple tof platform: Massive identification of proteins by LC-MS/MS methods (DDA Method using a Triple TOF) Differential quantitative proteomics using "label free" SWATH-MS. Targetted proteomics using SRM or MRM method. Posttranslational Modifications (PTMs): phosphorylation, glycosylation, glycation...usind softwares as PEAKS after DDA sample analysis.

Proteomics using 1D/2D-PAGE: Protein separation by means of one-dimensional electrophoresis (SDS-PAGE) ·Protein separation by Bidimensional Electrophoresis (2-DE) ·Differential proteomics using metabolic or chemical stable isotope labeling: 2D-DIGE, ITRAQ, SILAC…Posttranslational Modifications (PTMs): phosphorylation, glycosylation, glycation, etc.

The IDIS Epigenomics Unit was created with the aim to provide support to research groups and industry at national and international level to perform epigenetic studies in multiple scientific fields, at the level of specific genes and/or global epigenomics. For the epigenetic analysis carried out in the IDIS Epigenomics Unit, we have the most innovative technology in the field for epigenetic locus specific and/or epigenomic studies.

In addition to this technology the Epigenomics Unit have other accessory equipment (ProFlex PCR System from Applied Biosystems, equipment for electrophoresis analysis, and systems for DNA/RNA quantification such as Nanodrop, Qubit and Quantus). In addition we have a Pre-PCR and Post- PCR laboratory to perform the epigenetic and epigenomic analysis.

The IDIS Epigenomics Unit started its activity in 2018, providing a global support of the different steps in the epigenomic study for the discovery and validation of epigenetic biomarkers: i) Advice and help in the design of the study. ii) Preparation and processing of biological samples. iii) Epigenetic and/or epigenomic analysis with different types of methodologies. iv) Analysis, interpretation and information of the results. This Unit also has its own research lines, with a translational orientation in the epigenomics of endocrine diseases and nutrition, directed by Ana B. Crujeiras (PhD), and the epigenomics of cancer, directed by Angel Díaz-Lagares (PhD).

Using our infraestructures and the previous methodologies described we can perform the following epigenomic and epigenetic locus-specific analysis:

• NGS
• Microarrays for methylation analysis
• Pyrosequencing
• Quantitative methylation specific PCR (qMSP)
• Methylation specific PCR (MSP)

The Epigenomics Unit from IDIS will add value to any preclinical or clinical project that want to study epigenetic biomarkers involved in the development of different types of diseases. These studies can be both small-scale, to study epigenetic marks on individual genes or gene panels, and on a large scale, to perform a broader analysis of the epigenome. These epigenetic analysis will help identify or evaluate epigenetic biomarkers with clinical utility in different types of samples: frozen tissues, paraffins and biological fluids, as blood samples (liquid biopsy).

• Epigenomics using NextSeq500 (Illumina) for next generation sequencing (NGS): This service provides the possibility of studying epigenetic marks distributed along the genome. By means of our NextSeq500 system the epigenetic marks can be analyzed in part or in the whole genome of different types of samples.
• Epigenomics using HiScan Microarray System (Illumina): By means of this service we can provide information about the methylation status of multiple CpGs distributed throughout the genome in different types of samples. Using the Infinium MethylationEPIC Kit (Illumina) this system allows the methylation level analysis of more than 850.000 CpGs dsitributed in multiple locations of the genome.
• Epigenomics using PyroMark Q24 Pyro-Sequencer (Qiagen): This service allows the analysis of DNA epigenetic marks using the the PyroMark Q24 Pyro-Sequencer. This system provides quantitative information about the status of DNA epigenetic marks in different types of samples by means of a pyrosequencing approach.
• Epigenomics using StepOne Real-Time PCR (Applied Biosystems): We can analyze epigenetic marks using real time PCR methods. This methodology is very sensitive and can be applied to multiple samples, including biological fluids (liquid biopsy).

The following service will be available soon: Epigenomics using Droplet Digital PCR system (QX200, Bio-Rad): This Service provides the possibility to analyze epigenetic marks using droplet digital PCR. This is an ultrasensitive approach that can analyze epigenetic marks in very low amount of sample. It is possible to use this methodology in different types of samples, including biological fluids (liquid biopsy).