REPORT INTERNATIONAL:TB RICC BIOMARKER – JUNIOR INVESTIGATOR TRAINING

By: Gustiani Salim

The final session of the Junior Investigator training took place at Dr. Padmini Salgame’s Laboratory, located in the International Center for Public Health (ICPH), Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Newark, NJ, USA. This training continued to focus on discovering transcriptomic signatures for tuberculosis, using the advanced nanostring technology to study gene expression and identify potential biomarker candidates.

The wetlab training covered hands-on activities such as extracting RNA from blood samples collected in PAXgene RNA tubes, hybridizing RNA samples, loading samples into cartridges, and operating the nanostring machine. Unfortunately, the participants had to use dummy cartridges from the training kit instead of real cartridges, as the or-dered cartridges were unavailable during the training. Despite this, participants gained a clear understanding of the process. Additionally, the facility provided training on analyzing nanostring data, including data cleaning, quality control (QC), and visualization for presentation purposes.

Nanostring Technology for Gene Expression Study

Unlike the Fluidigm Biomark© platform, which primarily uses RT-qPCR methods, nanostring is a nucleic acid hybridization technique that allows for simultaneously examining up to 800 targets from 12 samples. Nanostring can be seen as a variation of the microarray method, but with the advantage of hybridizing directly with RNA samples, eliminating the need for reverse transcription, library preparation, or amplification that could introduce bias. Another benefit is that the nanostring system can work with various types of nucleic acid samples, including blood fluids, cell lysates, fresh tissues, and FFPE sections.

Participants learned to manually isolate total RNA from blood samples preserved in PAXgene Blood RNA Tubes (BRT) using the PAXgene® Blood RNA kit by PreAnalytiX (Qiagen). BRT contains an RNA stabilization reagent that protects RNA molecules from degradation and minimizes ex vivo changes in gene expression. The PAXgene® Blood RNA kit is column-based RNA isolation. After the lysis and protein digestion process, RNA selectively binds to the silica membrane as it passes through the spin column.

The quantitation of mRNA transcript expression was conducted using the Nanostring nCounter® SPRINT profiler instrument available in Dr. Padmini Salgame’s laboratory. Nanostring technology provides digital, multiplexed measurements of gene expression, offering simultaneous counts of hundreds of mRNA transcripts following solution-based hybridization with target-specific probes. The system utilizes three main components:

1. Capture probe: A 50 base pairs single-stranded sequence complementary to the mRNA target, conjugated with biotin.
2. Reporter probe: A single-stranded sequence complementary to the mRNA target, labeled with a fluorescent barcode specific to each mRNA target. The target-specific reporter and capture probes are collectively referred to as a CodeSet.
3. Cartridge: A microfluidic chamber system containing a streptavidin-coated imaging surface.

During hybridization, the capture probe and re-porter probe bind to the target mRNA at adjacent positions, forming an mRNA-probe complex (Figure 2).

When hybridized samples are loaded into a cartridge and run in the nCounter instrument, the fluidic processing system removes excess probes, un-bound targets, and other extraneous material. Purified target mRNA-probe complexes are deposited onto a streptavidin-coated imaging surface and immobilized via the biotinylated capture probe. Immobilized reporters are aligned, stretched, and immobilized again to create parallel fluorescent barcodes that can be imaged. An automated fluorescence microscope in the Digital Analyzer scans the cartridge, identifying each target molecule of interest. The number of detected fluorescence signals reflects the amount of a specific mRNA target in the sample.

The general steps of sample processing with the Nanostring nCounter for gene expression are summarized in the chart on page 13).

The nCounter SPRINT Profilers operate a multi-channel epifluorescence scanner and digital analysis system, designed to image samples processed within cartridges. The digital analysis system collects data by taking images and counting the number of each fluorescent reporter tag, tabulated as CSV files. There are two types of files generated by the system:

1. Reporter Library File: Unique to each custom CodeSet, containing information used during image processing to assign target identities to the barcodes.
2. Reporter Code Count File: Contains data for one of the twelve flow cells (assays) in a cartridge, detailing the counts for each target in an assay.

For analyzing and visualizing Nanostring data to measure the fold difference in gene expression, trainees received training on quality evaluation (QC/normalization) of nCounter data, basic statistical outputs, and publication-quality figures, using the following software options:

• nSolver analysis software: An integrated analysis platform for basic and advanced analysis.
• ROSALIND Platform: A cloud-based software platform for data visualization and pathway enrichment.
• R software packages for differential expression.

Public Health Research Institute Facility

Dr. Padmini Salgame’s laboratory is part of the Rutgers Regional Biocontainment Laboratory (RBL) under the Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Newark, NJ, USA. PHRI’s mission is to help eliminate infectious diseases worldwide through fundamental and translational research. The RBL is a specialized facility for safely and efficiently studying infectious diseases with highly transmissible agents. Located in the International Center for Public Health, participants were given a tour of the RBL’s facilities, including the most advanced laboratories for studying biological agents under biosafety level 3 (BSL 3) conditions. Additionally, the building houses an animal facility laboratory to accommodate research using trial animals such as mice and rabbits.

TB RiCC BIOMARKERS – JUNIOR INVESTIGATOR TRAINING, AN INSPIRING PROGRAM

Being selected as one of the participants for the TB RiCC Biomarkers – Junior Investigator Training was an invaluable opportunity. Participants learned directly from the world’s top experts in TB research. With only five participants, each received hands-on laboratory training, making the program highly effective and efficient. The training covered not only lab work but also the upstream and downstream processes conducted in RePORT member countries for TB biomarker research. Participants visited clinical sites to learn about patient recruitment, specimen collection, storage, examination, and data analysis, culminating in presenting results for publication.

Despite using advanced lab equipment unavailable in their home countries, participants gained essential insights and skills to apply in developing TB research or other infectious diseases. The diverse backgrounds and the extended training period fostered close relationships among participants, allowing them to exchange ideas and experiences from their respective countries. They learned to understand the challenges and solutions in TB research under the RePORT consortium. It is hoped that participants will continue to communicate and collaborate to advance TB research and the RePORT consortium.