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Fig. 1 | BMC Biotechnology

Fig. 1

From: Quantifying 35 transcripts in a single tube: model-based calibration of the GeXP multiplex RT-PCR assay

Fig. 1

Schematic representation of the GeXP multiplex RT-PCR assay for quantification of relative abundances of transcripts. a The reverse transcription of a RNA is primed with a gene-specific reverse primer (GSrev) that carries a universal priming site at its 5′-end to obtain a first strand cDNA fused with the universal priming site. The synthesis of the second strand cDNA is primed with a gene-specific forward primer (GSfwd) that also carries a yet different sequence as universal priming site. Because of the two universal priming sites incorporated, the cDNA can be amplified with universal forward and reverse PCR primers (Ufwd, Urev, respectively) independently of the gene-specific sequence. As the universal forward primer is labelled with a fluorescence tag at its 5′-end, the PCR product can be detected after capillary electrophoretic separation. b Gene-specific primers are designed to yield PCR products of different lengths that can be separated by capillary electrophoresis from each other and quantified through the area of individual peaks in the elution profile (c). c The gene expression pattern of different samples can be compared through the cDNA amplified from a spike-in RNA that serves as an internal standard. A DNA ladder (size marker) added to the cDNA sample prior to capillary electrophoresis allows to determine the size of the PCR products

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