Outline of the quantitative multiplex single-cell RT-PCR. Single-cell mRNA is retrotranscribed using a 3′-specific primer for each individual gene of interest (dark gray box). Next, single-cell cDNA is amplified on a first multiplex PCR where both 3′ (dark gray box) and 5′ (white box) primers of all different genes are present (15 cycles). Products of the first amplification are next split for a second seminested real-time PCR where a nested 5′ primer (light gray box) and the 3′ primer (dark gray box) are used to amplify each gene separately. This second round of amplification allows a precise quantification because test samples are compared with an RNA standard submitted to the same RT and amplification protocol.
