A typical QPCR biodistribution study assesses the presence of gene therapy vectors in a panel of tissues collected at multiple time points ranging from a few days to several months post administration. The panel of tissues typically includes blood, heart, brain, liver, kidney, bone marrow, ovaries/testes, lung, draining lymph nodes, spleen, and muscle samples at the site of administration.
- Sample received and assigned unique accession number(s)
- In-process samples also assigned unique tracking number(s)
- All samples stored in freezers with temperatures continuously monitored
- Extraction method validated for each tissue with high-spike, low-spike, and no-spike controls
- Target specific assay developed and optimized with and validated per ICH guidelines
- Tissues processed with controlled procedures designed to prevent cross contamination
- Nucleic acids isolated in designated Biosafety Level II extraction laboratory
- Nucleic acid concentration determined with Nanodrop
- Separate clean rooms free of nucleic acids used to prepare master mixes and negative controls
- Separate template room used to handle sample and positive controls
- PCR reactions performed on validated ABI 7900HT platform
- Standard curve generated to determine copy numbers
Below are typical characteristics of Avance Biosciences’ qPCR biodistribution study services. Actual numbers may vary and will be derived from custom validation.
- Limit of Detection: 5-10 copies of vector/1 μg of genomic DNA
- Sensitivity: 10-100 copies of vector/1 μg of genomic DNA
- Linearity range: 10 to > 107 copies/1 μg of genomic DNA
- Accuracy: ± 0.25 log10
- Precision: ≤10% CV
- Specificity: Positive and Negative controls will be tested
- Normalized target quantity are reported as the number of vector copies detected per genome detected
- As low as 50 copies per reaction is routinely detected
- Report reviewed by QA unit and signed by QA representative and study director
- Final report issued including: description of assay, results of the quantification, and tabulation of data
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