I extracted RNA from pellets of mammalian cell culture and turned it into cDNA. To determine the level of gene expression, I'll use qPCR. Should I first run the cDNAs through agarose gel electrophoresis? What outcomes should I expect when I use it?
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i would suggest to run a normal PCR using any reference genes such as beta actin, alpha tubulin and GAPDH. It is better to use the qPCR-complied primers (with the intro-exon boundaries) or I would say that they could be reference genes that will be used in your real time-PCR experiment. The presence of the PCR product from agarose gel electrophoresis would give you hint of your cDNA synthesis outcome. No template, no PCR! I hope this helps. Good luck!
Not at all! Don't spend your energy on performing a cDNA electrophoresis. Instead if you have checked the integrity of your RNA and if it was good then you shouldn't worry. Since you are concerned about your qPCR data, I would recommend you to start with atleast three-four different housekeeping genes and check which one fits the best for you.
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Agarose gel electrophoresis for cDNA would be unnecessary if you have checked the RNA integrity in denaturing agarose.
Agarose gel electrophoresis is not typically required for the cDNA control process in gene expression analysis using qPCR. The purpose of agarose gel electrophoresis is to assess the integrity and quality of RNA samples before cDNA synthesis. However, once cDNA is synthesized from the RNA, running it on an agarose gel is not necessary for qPCR analysis.
In gene expression analysis using qPCR, the cDNA is used as the template for amplification of specific target genes. The qPCR assay itself provides information about the relative expression levels of the target genes. The amplification curves and cycle threshold (Ct) values obtained from qPCR are the primary indicators of gene expression levels.
Agarose gel electrophoresis is more commonly used to assess the quality of RNA samples before cDNA synthesis. It helps to ensure that the RNA is intact, free from degradation, and of sufficient quality for downstream applications. By visualizing the RNA bands on the gel, you can confirm the presence of intact 18S and 28S ribosomal RNA (rRNA) bands as well as the absence of degradation products.
Therefore, for your specific purpose of gene expression analysis using qPCR, agarose gel electrophoresis of cDNA is not required. The outcomes to expect when using qPCR for gene expression analysis include amplification plots, melting curves, and quantification cycle (Cq) values, which can be used to calculate relative gene expression levels.
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