Olink Reveal multiplex protein analysis

Method for multiplex protein biomarker analysis (>1000 proteins) with sequencing readout using the Illumina NovaSeq platform.

As a joint effort between the SciLifeLab National Genomics Infrastructure (NGI) and the Affinity Proteomics Uppsala unit (APU) at SciLifeLab Proteomics platform, the Olink and Olink Reveal assays are available in our services. The assays are based on the Proximity Extension Assay (PEA) but unlike the qPCR 24 to 92-plex Target assay, the Explore and Reveal assays utilizes high throughput sequencing as readout.

Below you can find some basic information regarding sample requirements and data output for the Reveal assay. If you have any further questions, please contact us at explorelab@scilifelab.uu.se for more information about our service, or visit Olink´s website, Olink Reveal – Olink.

Sample Requirements

• Sample type: Plasma, serum or CSF. Other sample types are also applicable, please contact us to discuss what is feasible. NB! All samples in a project must be of the same type, even the same plasma type is required.
• Number of samples: 1×86 samples or multiples thereof.
• Volume of sample needed:
  • ≥25 µL (max 80µl)
• Plate design:
  Each plate is designed to include 86 samples and 10 controls. The samples must be placed in the wells A1-F11 according to the template below. The last wells (G11-H11 and A12-H12) is reserved for the controls and must be left empty. All controls are provided by the facility.
  Please make sure to use a 96-well plate and seal that are suitable for storage at  -80°C. The plate should preferably have V-shaped wells. We recommend the ABgene, AB-0800-L plate and ABgene, AB-0558 sealer. Plates and sealers can be provided upon request.

• Randomization:
  Samples must be randomized within and between the plates included in the project. This in order to minimize the risk of removing true biological differences during QC and to be able to check for row and column effects..

Description of the method

The PEA technology is based on two matching antibodies binding to a protein, upon binding, the DNA tags from the two antibodies come into contact/proximity and the DNA tags hybridize and are extended making a unique DNA barcode for each protein analyzed. After amplification, digital DNA counting is performed using sequencing and the resulting counts directly relate back to the concentration of the protein in the sample. We evaluate the quality of the sequencing library prior to sequencing. The sequencing will be carried out the specific setup for the application as stated in the agreement.

Data processing, quality control and delivery

During the sequencing run, known sequences are read and the BCL files generated are subsequently converted to counts files. The counts directly represent the protein concentration in the sample analyzed.

The Counts file is processed in Olink proprietary software NPX Map to generate NPX (Normalized Protein eXpression) values. During this process an extensive quality control is performed where the control samples and markers are evaluated to rule out bad assay performance and variation between plates. The quality control samples are also used for normalization. Data from the Olink Reveal assay is delivered as NPX values and accompanied by a Certificate of Analysis describing the project and data. The data is delivered encrypted using CryptShare.

Last Updated: 7th April 2025