Simultaneous Isolation and Parallel Analysis of Genomic DNA and Total RNA: A Comprehensive Approach

Adeno-associated vectors (AAVs) play a crucial role in delivering transgenes into host cells, making them indispensable for both preclinical and clinical research in gene therapy. The efficiency of this process hinges on the vector copy number (VCN) and transgene expression. VCN is quantified by analyzing genomic DNA (gDNA) from bulk cells or tissue using qPCR or digital PCR (dPCR), while transgene expression is assessed by identifying and quantifying RNA. The conventional approach to total nucleic acid extraction involves dividing a single tissue sample into two parts, with one part used to isolate DNA and the other to isolate RNA. This may potentially lead to less accurate VCN or transgene expression assessments, particularly in heterogeneous tissues such as the brain, where cellular subtypes can vary significantly between sample punches. Applying a novel approach using solid phase reversible immobilization (SPRI) bead-based technology, we were able to sequentially and selectively bind DNA and RNA to the beads. Our findings show that the gDNA and RNA yield from various tissue types exceeded the minimum yield requirement, achieving a 100% pass rate. Furthermore, the results of the extraction cross-contamination run indicated the absence of contamination between wells, as evidenced by the DNA yield from negative control samples randomly distributed across the samples.