D.1 SOW.pdf

Version 11-30-2016-1 FAR Part 11—Describing Agency Needs

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

RNAScope analysis of human pluripotent stem cells derived nociceptors

Statement of Need and Purpose:

The Stem Cell Translation Laboratory (SCTL) requires the services of Advanced Cell Diagnostics in order to meet project milestones. RNAscope is a novel proprietary RNA in situ hybridization (ISH) method that enables routine detection and visualization of virtually any expressed gene in any tissue such as cultured cells, FFPE, Fresh frozen and fixed-frozen tissue.

This service will be utilized by the SCTL to analyze the subpopulations of human pluripotent stem cells derived nociceptors.

Background Information and Objective:

The SCTL has recently developed a protocol for differentiating hPSC into nociceptors. We are particularly interested in the subpopulations in our nociceptor cultures. We plan to evaluate expression of thirty‐six (36) mRNAs across eight (8) four‐plex and two (2) three‐plex RNAscope fluorescent panels in one (1) human iPSC‐derived nociceptor sample in order to validate single cell RNAseq data and to determine the cell types present.

Period of Performance:

The period of performance shall be 16 weeks from the receipt of order (ARO) including the receipt of the samples intended for sequencing. The Government anticipates, but it does not guarantee, the shipment of 1 (one) full set of samples within 30 days, already included in the Period of Performance

SCOPE OF WORK

General Requirements:

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:

The SCTL will culture the cells and fix the cells. The fixed chamber slides will then be shipped to Advanced Cell Diagnostics as one full set. Advanced Cell Diagnostics will perform RNAscope Multiplex Fluorescent ISH assay for thirty‐six (36) mRNAs in one (1) human iPSC‐derived neural cell sample and perform positive and negative control assays to assess RNA quality and to optimize assay conditions for the sample set, followed by performance of target assays on the samples that pass QC.

Specific Requirements:

Sample QC and Optimization

Samples shall be evaluated first for RNA quality using a positive (low copy housekeeping gene) and a negative (non‐specific bacterial gene) control probe. Based on qualitative evaluation, additional optimization of sample treatment may be necessary and require additional sample slides, if available. Visual semiquantitative scoring shall be performed on control probe staining to assess QC pass/fail.

RNAscope Fluorescent 3‐plex and 4‐plex Marker Assays

ACD shall use the optimized assay conditions established in Phase I to perform ISH assays to investigate the expression of marker RNAs in the QC’d sample.

Slide scanning/Image acquisition

40x fluorescent full‐slide scanning shall be attempted on 3D‐Histech scanner. It is uncertain whether it will be possible to scan the stained chambered slides. Attempts shall be made with assistance from 3D Histech prior to scanning stained slides from this study. In the event that the stained chambered slides cannot be scanned, then representative, field‐of‐view images shall be captured using a Nuance™ (PerkinElmer) multispectral imaging microscope.

Scoring and Data Analysis

Quantitative image analysis shall be performed with HALO software in each sample ‐ Eight (8) chambers after 4‐plex and two (2) after 3‐plex assays. Quantification endpoints will be acquired using a probes/cell methodology that associates any probe signal with the corresponding cell.

Reported data will include (1) total number of cells per ROI, (2) average number of dots per cell,

(3) calculated H‐Score based on binned categories of expression, (4) percentage of cells positive (%+), (5) percentage of cells dual‐positive (%+,+), (6) percentage of cells triple‐positive

(%+,+,+), and (7) percentage of cells fourplex‐positive (%+,+,+,+) for the markers.

GOVERNMENT RESPONSIBILITIES

The SCTL will culture the human iPSCs derived nociceptors and provide the vendor with the fixed cells on chamber slides.

DELIVERY OR DELIVERABLES

Results will be provided in a Study Brief including the staining results and HALO analysis describing materials and methods as well as final assay conditions used for the sample set. A small sample of representative images will be included.

The study shall also include stained slides of positive, negative, and target probes, whole slide images (WSI) of all slides scanned at 40x magnification provided on an external drive, final optimized assay conditions, and a report detailing materials and methods, results, and conclusions

OTHER CONSIDERATIONS

Travel:

The contractor shall not be required to travel to perform these requirements

Data Rights:

FAR Clause 52.227-14, Rights in Data-General (May 2014) is included in the solicitation and resultant award. Under 52.227-14(c)(1), the contractor must obtain approval from the contracting officer to assert copyright in any data first produced in performance of this contract and published in academic, technical or professional journals, symposia proceedings, or similar works. Under 52.227-14(d)(2), the contractor must obtain approval from the contracting officer to use, release to others, reproduce, distribute, or publish any data first produced or specifically used by the contractor in the performance of this contract.

In addition, the contractor will keep the results of the acquisition confidential.