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Figure 3 | BMC Molecular Biology

Figure 3

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

Figure 3

Specificity of the SYBR green I assays for cod follicle-stimulating hormone beta subunit ( FSHβ ), luteinizing hormone beta subunit ( LHβ ), and elongation factor 1 α ( EF1α ). Specific primers for cod FSHβ, LHβ, and EF1α mRNA were tested and validated. A Melting curve plotted as the negative change in fluorescence per time as a function of temperature. The specific melting peak for each qPCR product in the melting curve analysis indicates that only one product has been amplified. The specific melting temperature (Tm) was for FSHβ 84°C, LHβ 88°C, and EF1α 86°C. Non-template controls (NTC) performed by substituting cDNA with nuclease-free water as PCR template showed the absence of primer dimers or other interfering factors. Also, substituting cDNA with genomic DNA as PCR template showed no amplified product, confirming that genomic DNA did not interfere with the PCR results. B qPCR products visualized on a 2% agarose gel stained with EtBr. Lane 1: 50 bp molecular ladder. Lane 2: reverse transcribed pituitary RNA and specific primer pair for FSHβ showing one band with the expected size 63 bp. Lane 3: reverse transcribed pituitary RNA and specific primer pair for LHβ showing one band with the expected size 81bp. Lane 4: reverse transcribed pituitary RNA and specific primer pair for EF1α showing one band with the expected size 100 bp. Lane 6-8: NTC for FSHβ, LHβ, and EF1α, respectively, confirming the absence of primer dimers and/or other interfering factors in the qPCR reaction.

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