Journal: PLOS ONE

Article Title: Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

doi: 10.1371/journal.pone.0280242

Figure Lengend Snippet: DNase I reactions were prepared that comprised RPP30 gBlock and AAV2 with different additives. Samples were incubated at 37°C for 30 min and then serially diluted into the ddPCR concentration range with polyA buffer. Capsids were lysed at 95°C for 10 min prior to assembling ddPCR reactions. (A) RPP30 concentration without and with DNase I. The estimated RPP30 concentration for samples containing DNase I is indicated with numerical values because the bars are not visible. (B) eGFP concentration with DNase I digestion. Error bars represent the 95% confidence interval.

Article Snippet: Two separate DNase I (NEB, M0303L) experiments were performed with a purified AAV2 vector formulation.

Techniques: Incubation, Concentration Assay

Journal: PLOS ONE

Article Title: Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

doi: 10.1371/journal.pone.0280242

Figure Lengend Snippet: Seven different dilution buffer formulations were used to dilute AAV2 after DNase I digestion. Buffer identities are listed in . Buffers 1‒3 are based on DNA suspension buffer and buffers 4‒7 are based on PCR buffer. For reference, buffer 1 is polyA buffer and buffer 7 is PCR buffer. Duplex reactions with ITR2 FAM and eGFP HEX were analyzed. (A) The concentration of the ITR (black bars) and eGFP (gray bars). (B) The concentration ratio of ITR2 to eGFP. The error bars represent the 95% confidence interval.

Article Snippet: Two separate DNase I (NEB, M0303L) experiments were performed with a purified AAV2 vector formulation.

Techniques: Concentration Assay

Journal: PLOS ONE

Article Title: Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

doi: 10.1371/journal.pone.0280242

Figure Lengend Snippet: PolyA buffer and PBS with 100 ng/μL polyadenylic acid (PBS + pA) were prepared with Pluronic F-68 concentrations that varied from 0–0.1% and used to dilute AAV2 after DNase I digestion. Duplex reactions with ITR2 FAM and eGFP HEX were analyzed. (A) The concentration of the ITR (black bars) and eGFP (gray bars). (B) The concentration ratio of ITR2 to eGFP. The error bars represent the 95% confidence interval.

Article Snippet: Two separate DNase I (NEB, M0303L) experiments were performed with a purified AAV2 vector formulation.

Techniques: Concentration Assay

Journal: PLOS ONE

Article Title: Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

doi: 10.1371/journal.pone.0280242

Figure Lengend Snippet: After DNase I digestion, viruses were serially diluted using polyA+ buffer into the ddPCR concentration range. The viral samples were either lysed at 95°C for 10 min (pre-droplet) or added directly (in-droplet) to ddPCR reactions and lysed using the polymerase activation step of the PCR thermal cycle. The (A) eGFP concentration and (B) ITR2/eGFP concentration ratio. The error bars represent the 95% confidence interval.

Article Snippet: Two separate DNase I (NEB, M0303L) experiments were performed with a purified AAV2 vector formulation.

Techniques: Concentration Assay, Activation Assay

Journal: PLOS ONE

Article Title: Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

doi: 10.1371/journal.pone.0280242

Figure Lengend Snippet: After DNase I digestion, AAV5 was incubated either without (no PK) or with (PK) proteinase K for 2 hours at 50°C and then further processed as described in the Materials and Methods. The two samples were serially diluted in parallel with polyA+ buffer and either added directly to the ddPCR supermix (no capsid lysis step) or had the capsid thermally lysed (with capsid lysis step) prior to adding to the ddPCR supermix. The ITR2 concentration (black bars) and eGFP concentration (gray bars) is shown. The values indicate the ITR2/eGFP concentration ratio. The error bars and numerical errors represent the 95% confidence interval.

Article Snippet: Two separate DNase I (NEB, M0303L) experiments were performed with a purified AAV2 vector formulation.

Techniques: Incubation, Lysis, Concentration Assay

Journal: PLOS ONE

Article Title: Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

doi: 10.1371/journal.pone.0280242

Figure Lengend Snippet: An AAV2 sample was DNase I treated and lysed for 10 min at 95°C prior to droplet formation (pre-droplet lysis) and then used as a template for duplex ddPCR reactions using CMV-Enh FAM and the indicated HEX assay with either no enzyme, MspI, MseI, or a double digest with MspI and MseI. The (A) calculated linkage percentage and (B) concentration ratio of CMV-Enh FAM to the HEX assay with the corresponding standard deviations are shown.

Article Snippet: Two separate DNase I (NEB, M0303L) experiments were performed with a purified AAV2 vector formulation.

Techniques: Lysis, Concentration Assay