(A) Quantification of RCA–RCA amplification-fold in genomic DNA. QRT-PCR was performed either directly from unamplified BT474 genomic DNA or from the RCA–RCA-amplified product for three single copy genes. Curves 1–3 GAPDH, IL9R, and CYC respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6 GAPDH, IL9R, and CYC respectively, using unamplified DNA as starting material. (B) Determining the lower limit of starting material for which RCA–RCA is possible. A comparison of QRT-PCR for HER2 performed directly from unamplified BT474 genomic DNA or from the RCA–RCA-amplified product is depicted, following serial dilution of the DNA. Curves 1–3: 0.3, 0.03, and 0.003 ng respectively, using RCA–RCA-amplified DNA as starting material. Curves 4–6: 0.3, 0.03, and 0.003 ng respectively, using unamplified DNA as starting material. HER2 is amplified in BT474 genomic DNA and 3 pg genomic DNA (curves 3 and 6) are expected to contain ∼15–30 HER2 molecules.
