Figure 1. Overview of the qualitative multiplex single-cell RT-PCR and Primer Specificity.

A) Single cells are sorted into individual wells in a 96 well plate. Binding of the gene specific 3′ primer to the mRNA allows reversed transcription to cDNA. This is then followed by 22 cycles of multiplex pre-amplification. The 1^st round products are diluted 1/5 and then split for the 2^nd round real-time amplification with nested 5′ and 3′ primers, and specific amplification was detected with the addition of specific LNA probes (F = FAM, Q = Quencher). B) Specificity of all 11 TF and ß-actin (positive signal control) was examined by electrophoresis in 1.5% Agarose gel, to determine correct sized amplicons (for amplicon sizes refer to Supplementary). Real-time PCR was subsequently used to confirm specificity by checking single melt peaks using SYBR®-green I.