AAV Titration by qPCR

Assemble all required equipment including pipettes, tips, plates, master mix, primers, standards, and AAV reference samples. Keep critical reagents on ice and ensure DNase treatment of AAV samples to remove contaminating plasmid DNA.

Set up serial dilutions by adding water to wells using a multi-channel pipette, then sequentially transfer and mix samples across rows according to the dilution scheme. Continue until reaching the final dilution row.

Load 5 microliters of selected dilutions in duplicate onto the qPCR plate using a multi-channel pipette. Include 5 microliters of plasmid standards in duplicate and add a no-template control well.

Prepare the qPCR master mix by combining water, SYBR Green master mix, and forward and reverse primers in the specified ratios. Distribute 15 microliters of master mix to each well using a multi-channel pipette and mix thoroughly.

Seal the qPCR plate with transparent adhesive film and briefly spin down. Run the plate on the qPCR instrument using the SYBR Green detection protocol.

Confirm the baseline setting is appropriate for background removal. Verify that the standard curve has an R-squared value near 1.0 and amplification efficiency around 100%, with approximately 3.3 CT difference between tenfold dilutions.

Examine the melt curve for a single peak indicating specific amplification without primer dimers. Confirm sample duplicates are within 0.5 CT range and no-template controls show higher CT values than all standards.

Use the qPCR software to calculate the number of AAV genome copies per milliliter based on the standard curve, accounting for all dilution factors and volumes loaded.