TITLE

RNA Biomarkers for predicting radiation injury for radiation biodosimetry.

BACKGROUND

Radiological attacks and nuclear detonations can cause mass casualties. Victims may have received substantial radiation doses and may not immediately exhibit visible symptoms of radiation sickness. Victims with whole body or substantial partial body exposure >2 Gy will require immediate treatment within 24 hours to mitigate radiation injury while others will require both intermediate and long-term management for possible injury to the marrow, gastrointestinal tract, lung and other organs. Early prediction of possible acute intermediate and delayed effects will enable timely therapeutic interventions which will not only reduce death, but also improve the quality of life for the victims.

The NCI needs to identify radiation-induced global miRNA/mRNA/lncRNA expression patterns after different doses and time points following partial body irradiation in a non-human primate model. A specific functional miRNA expression signature previously derived from mouse lung tissue after whole body irradiation from the NCI laboratory will be assessed in the whole blood and serum samples after partial body irradiation in these non-human primate model and also using a human organ on a chip model. These could then be developed as part of the Point of Care and High Through-put screening platforms that are under development by other groups as part of NIAID and BARDA programs.

OBJECTIVE

The primary objective of the project is to obtain microRNA, long non-coding RNA, and mRNA expression information by performing RNAseq analysis with the following workflow:

1. Preparation of cDNA libraries
2. RNA QC before and after library prepration
3. Performing RNA seq on Illumina platform
4. Obtaining 20 million reads/sample
5. Aligning the reads to reference genome for data analysis 

With this data, the NCI will identify biomarker signatures of radiation exposure and of organ-specific radiation injury.

SCOPE

This project is a multi-step process with each step dependent on the successful completion of the former. The process starts with a quality check of the RNA and follows with sequential steps of rRNA depletion, globin RNA depletion, adapter ligation, cDNA preparation, sample purification and QC, reading on an Illumina platform, aligning reads to a reference genome, and finally data clean-up and analysis for identifying differentially expressed coding and non-coding RNAs.

CONTRACT REQUIREMENTS/ AND PERSONNEL QUALIFICATIONS

The contractor shall perform the following tasks:

1. 1. mRNA and lncRNA sequencing
      1. Total RNA sample quality control
         1. Nanodrop: tests RNA purity
         2. Agarose Gel Electrophoresis: tests RNA degradation and potential contamination
         3. Agilent 2100: checks RNA integrity
      2. Library Construction
         1. rRNA removal
         2. Fragmentation
         3. cDNA synthesis
         4. Adapter ligation
         5. Size selection
         6. PCR enrichment
      3. Library Quality Control
         1. Qubit 2.0 fluorometer
         2. Agilent 2100
         3. Quantitative PCR
      4. Sequencing using Illumina platforms
      5. Data Analaysis
         1. Quality control
         2. Read mapping to reference genome

1. 1. Small RNA sequencing (microRNA)
      1. Total RNA sample quality control
         1. Nanophotometer: tests RNA purity
         2. Agarose Gel Electrophoresis: tests RNA degradation and potential contamination
         3. Agilent 2100: checks RNA integrity
      2. Library Construction
         1. Library prep for small RNA sequencing using NEBNext Multiplex Small RNA Library Prep Set for Illumina
      3. Library Quality Control
         1. Agilent 2100 using DNA High Sensitivity Chip
      4. Clustering and Sequencing
         1. Clustering of index-coded samples on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumina)
         2. Sequencing of libraries on an Illumina platform to generate 50bp single-end reads
2. 1. Data Analysis
      1. 1. Quality control
         2. Data normalization
         3. statistically significant (FC ≥2 or ≤ 0.5; p-value <0.05) genes were identified from each animal and timepoint for further analysis
         4. Read mapping to reference genome.

TYPE OF ORDER

This will be issued as a Several Firm Fixed Price Purchase Order.

PERIOD OF PERFORMANCE

The period of performance shall be from March 1, 2021 to August 31, 2021.

PLACE OF PERFORMANCE:

Novogene, 126 Corporate Boulevard, South Plainfield, NJ 07080