Figure 2
From: Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol
Specificity of the SYBR green I assay for rat prolactin ( PRL ). A. Melting curve showing the negative change in fluorescence per time as a function of temperature. The specific peak in the melting curve indicates that the PCR has only amplified one product. The melting temperature (Tm) for the PRL amplicon was 83.0°C. The non-template control (NTC), confirms the absence of primer dimers or other interfering factors of the assay besides the cDNA template. Substituting cDNA with cell lysate where the reverse transcription step was omitted (÷RT) showed no amplified product, confirming that genomic DNA did not interfere with the PCR results. B qPCR products visualized on a 1% agarose gel stained with EtBr. Lane 1: 100 bp molecular ladder. Lane 2: reverse transcribed cell lysate and specific primers for PRL amplifying one product of the expected size (216 bp). Lane 3: specific primers for PRL tested on cell lysate without reverse transcription, confirming the primer pair's insensitivity for genomic DNA. Lane 4: NTC for PRL.