Gene expression analysis
RT-qPCR (Reverse transcription quantitative polymerase chain reaction) is the most common approach for quantitation of RNA levels, and this method is widely used in our laboratory.
The method is based on a combination of three steps: 1) The reverse transcription of RNA into complemantary DNA (cDNA), 2) amplification of cDNA of interest using PCR and 3) detection and quantitation of amplified PCR product at each PCR cycle using fluorescent reporter molecules.
We have used RT-qPCR to examine the expression pattern of selected genes in the Serca2 knockout (KO) mice. The figure shows an amplification plot for Serca2 of samples from the control group and the KO group 6 days after disruption of the serca2 gene. The control group, with relative higher expression of the Serca2 gene, creates a detectable PCR-product at an earlier time point (lower cycle number) than the KO group, confirming the downregulation of serca2 in the KO group.
Genotyping of knockout (KO) mice
Knockout (KO) mice are genetically modified (transgenic) mice in which one or more genes are turned off. Such animal models can be helpful tools in providing information of the biological function of a gene.
At the institute we use several mouse strains with different gene modifications. The genotypes of these mice are determined by PCR (polymerase chain reaction), a method used to amplify a specific region of a DNA strand. The PCR products are analysed by agarose gel electrophorersis to check for precence or not of DNA fragments of interest.
The figure shows results from genotyping of mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO) (K. Andersson, G. Christensen). The upper band represents Cre-recombinase wich is expressed in the KO only , the lower band represents an internal control, thus samples with two bands are from SERCA2 KO mice, samples with one band are from control mice.
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