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Figure 8 | BMC Molecular Biology

Figure 8

From: Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

Figure 8

Single-cell qPCR on rat (GH4) cells, using specific primer pair for rat prolactin ( PRL ). Directly following qPCR, the different samples were subjected to a melting curve analysis. The peak in the melting curve shows the presence of a rat PRL-specific amplicon in the sample containing a single cell. The melting temperature (Tm) was 83.0°C, the same as for the amplified product from 10 000 cells. The lack of peaks in the melting curve analysis from the collected extracellular solution confirms the absence of amplified nucleotide and thus contaminating factors in the extracellular solution harvested together with the cell. The reverse transcription (RT) step was also routinely excluded from samples containing a single cell to confirm the primer pair's insensitivity to genomic DNA. For each qPCR run, a non-template control (NTC) was used substituting the cDNA with nuclease-free water in the qPCR mixture. The NTC melting curve analysis detected no primer dimers or contaminations in the reaction mixture.

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