SOURCES SOUGHT
B -- LABORATORY TESTING
- Notice Date
- 4/29/2016
- Notice Type
- Sources Sought
- NAICS
- 541380
— Testing Laboratories
- Contracting Office
- Department of Health and Human Services, National Institutes of Health, National Institute of Child Health and Human Development, Contracts Management Branch, 6100 Executive Blvd., Suite 7A07, MSC7510, Bethesda, Maryland, 20892-7510
- ZIP Code
- 20892-7510
- Solicitation Number
- NICHD-16-052
- Archive Date
- 5/26/2016
- Point of Contact
- Patricia Haun, Phone: 301-443-7786
- E-Mail Address
-
haunp@mail.nih.gov
(haunp@mail.nih.gov)
- Small Business Set-Aside
- N/A
- Description
- The National Institutes of Health (NIH), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) is conducting a market survey to determine the availability and technical capability of qualified small business, veteran-owned small business, service-disabled veteran-owned small business or HUBZone small businesses that can provide hCPE ΔN Replication of Data. The NICHD DIR is seeking a scientific group to replicate a series of experiments to address the existence of hCPE ΔN, the human carboxypeptidase gene, and to provide data that replicates both published and of unpublished data. Recent evidence (unpublished) has suggested that the CPE deltaN splice variant is an artifact of the reverse transcription (RT) step and/or the PCR step leading to the possibility that hCPE deltaN may not exist naturally but that it is generated from the WT hCPE sequence. The possible reason for this lies in the unusually high G/C content of the hCPE gene (~80-85%) where the deltaN deletion has been reported to occur in exon 1. This region may form complex secondary structures that would be difficult for polymerases to read through accurately using standard conditions. In addition this region (exon 1) has multiple micro-homology domains within it giving rise to possible RT and PCR artifacts. Also, the 40 kDa hCPE deltaN protein is not predicted to be generated from the mRNA transcript. Applicants to undertake this project will have expertise in cancer genetics research and a track record of expertise and publications in the areas of cancer metastasis. The scope of work will require bioinformatics analysis, quantitative RT-PCR, primer sequencing, and cloning, as well as Northern and Western blots. SCOPE OF WORK: Four specific aims are to be addressed: Specific Aims: 1.Demonstrate that the primers, described as specific for hCPE deltaN and used in Lee et al. (J Clinical Investigation, 2011), are specific for hCPE deltaN and cannot (or can) cross-prime the WT sequence. Bioinformatic analysis of the primer sequences should be included. As controls, reagents such as the plasmids containing the ORF of WT hCPE and the ORF of the proposed hCPE deltaN (and empty vector) are available from the NICHD. All reagents received should be sequenced to confirm their accuracy prior to use and used in serial dilutions as template for PCR or transfected into a cell line and analyzed by the quantitative RT-PCR procedure described in the original paper. 2.Clone any human CPE mRNA transcripts from hepatocellular carcinoma (HCC) tissue and/or MHCC97 cell lines (high and low metastatic phenotype, MHCC97H, MHCC97L) as these were reported to contain CPE deltaN in the original Lee at al. paper and recently in Huang et al., Tumor Biology, 2016; see also Tumor Biology, Zhou et al., 2013. For consistency and reproducibility, the colon cancer cell lines, SW480 and HT-29, should also be analyzed in parallel as they were reported in the Lee et al. paper (low and high metastatic phenotype). These cell lines can be obtained from NICHD. Use of the Ambion FirstChoice RLM-RACE kit (or equivalent) is preferred because it favors transcripts with a 5'CAP, indicative of a naturally occurring mature RNA transcript. Touch-down PCR should be employed to minimize artifacts. It is expected that standard PCR cycle numbers will be used in the 5'-RACE cloning, but to test for the presence of rare transcripts, increased cycle numbers and/or nested PCR may be appropriate. If required, however, the relevance of the rarity should be noted. Sequence all products cloned using high stringency for the sequencing procedure because the G/C content in this region is very high and may cause mis-reads. 3.Perform Western blot analysis on HCC cancer tissue and/or MHCC97L and MHCC97H cell lysates for the detection of any CPE protein. hCPE deltaN protein has been reported in these cells as a 40 kDa protein (Lee et al). Analysis should be on total soluble cell lysate (as in Lee at al.) and on nuclear versus cytosolic fractions. Use of an endocrine cell line that contains CPE should be used as a control for the extraction procedures and as a positive control for the Western blot, e.g., AtT20 cells, a mouse corticotroph cell line. At least two unique anti-CPE antisera should be used, i.e., antibodies made against different antigenic sources and made in two different hosts (rabbit, mouse, goat). It is not recommended to use the BD Bioscience monoclonal anti-CPE antibody, which can give non-specific bands at 40kDa. If used, it should be as a third antibody with all controls (omission of primary antibody, use of an absorption control where possible). Use of siRNA oligos transfections to reduce endogenous CPE is highly recommended to demonstrate specificity. Stealth siRNA oligos specific for hCPE are available from NICHD. 4.A Northern blot on HCC and CRC cell lines using standard procedures. A new semi-quantitative RT-PCR assay has been developed to identify and measure hCPE deltaN in cell lines and cancer tissues (see Appendix III for primer sequence and cycle conditions). An independent bioinformatic analysis of the primer sequences should be included in the report. Using these primers and conditions, confirm the presence of hCPE deltaN in HCC tissue and/or MHCC97L and MHCC97H cells using standard RT-PCR molecular biology procedures. In parallel, perform an identical analysis using the following modifications that are incorporated to minimize artifacts of the RT. After denaturation of the RNA template at >65 C (in the presence of the random primers), the RNA is to be cooled directly to 50 C to prevent refolding and a thermostable RT enzyme used (optimum temperature ~50 C). In addition, to minimize artifacts of the PCR, perform the annealing temperature at 65 C instead of 60 C. Compare and contrast the different procedures to identify if bands are reduced or eliminated when more stringent conditions are used. Confirm the identity of each PCR product by sequencing (which will be provided at a later time). Interested firms with the capability of providing the service shall submit a capability statement to assist the Government in determining whether or not this procurement will be set-aside for any of the testing described above. This is in accordance with Federal Acquisition Regulation (FAR) 19.502-2(b). The intended procurement will be classified under North American Industrial Classification (NAICS) code 541380 with a size standard of $15.0M. All respondents are requested to identify their firm's size and type of business. Interested firms responding to this market survey must provide (a) capability statement demonstrating their experience, skills and capability to fulfill the Government's requirements for the above. The capability statement shall be in sufficient enough detail, but not to exceed 30 pages, so that the Government can determine the experience and capability of your firm to provide the requirements above. Your capability statement, not to exceed 30 pages, should include references. A copy of the capability statement must be received by email (Haunp@mail.nih.gov) no later than close of business (3:00p.m local time at designated location) May 11, 2016. Responses by fax WILL NOT BE ACCEPTED. This synopsis is for information and planning purposes and is not to be construed as a commitment by the Government, nor will the Government pay for information solicited.
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