Bioassay Development: RTqPCR and qPCR Assays

qPCR workflows have been a mainstay of early discovery research and clinical biomarker studies for decades. As a fully mature technology platform, virtually any gene of interest is available as a commercial reagent from multiple vendors. Additionally, applications have expanded into the cGxP arena as a means to evaluate residual nucleic acid levels in therapeutics as well as unwanted contaminants such as Mycoplasma. Reagent technology and instrumentation have advanced to provide robust, reliable and faster sequence detection and custom reagent development for unique targets is easily performed. Curia offers significant experience and capacity in custom reagent design, study design and evaluation of mRNA and gDNA targets utilizing Thermo-ABI TaqMan™ Reagents and Instrumentation.

Highlights:

• Significant team experience with Custom Probe/Primer Assay Design utilizing Thermo™ or IDT® reagents
• Multiple instruments: (2) Quantstudio 5s, Quantstudio 6 and Quantstudio 7 with Orbitor plate handler. All 384 well format utilizing Fast chemistry
• Relative ddCt Quantitation or Absolute Quantitation utlizing linearized plasmid standards or gDNA
• Simple workflows utilizing inventoried, commercial probe/primers and quick turnaround times
• Complex, multi-analyte, multiplexed workflows with 10-100 Genes of Interest (GOI) and 100s of samples.
• Development of residuals assays or titer assays for process fluids (VG for AAV/Lenti, Residual Host Cell DNA and RNA) and cGxP qualification/validation workflows.
• Virtually any sample input: Tissues, Cells, Biofluids and FFPE.

Process Development Case Study:

Development of qPCR-based Method for Determination of Residual DNA in IVT Process Fluids for an mRNA Therapeutic

• Purpose: Client’s process for generating RNA therapeutic carries residual plasmid DNA and genomic DNA through purification process. Accurate and specific methodology needed for assessing levels of contamination for process development efforts with the ultimate goal of transferring protocol to Client’s QA Group
• Design: Absolute quantitation with plasmid-based standard curves. Custom designed multiple probe and primer sets for client’s sequence of interest.
• Optimization Work: Validated probe sets and performed assessment of sample matrix influence on data output as well as spike/recovery tests with standard plasmids. (Amplification Plot and Standard shown below)
• Outcome: Recurrent support of assay for client ongoing and development of additional analogous assays for additional therapeutics

Please reach out to our team for additional information!