A particular gene (gene A) is reported to be downregulated in cancer cells as
compared to the normal cells. How can you prove this with the help of PCR?
Hello Bhaskar Chatterjee
In order to study gene expression by PCR you need to perform relative quantification by RT-qPCR. First you need to determine your target primer sequences whether or not these primers bind to exon-exon junctions otherwise you might end amplifying genomic DNA. Selection of candidate reference gene/genes for normalization is very crucial. They should have stable expression levels in your control as well as your experimental group. Various methods are available for calculation of relative gene expression like the Livak method, Pfaffl method depending on your assumption of your PCR efficiency. Please go through the MIQE guidelines which will give you a better understanding.
https://academic.oup.com/clinchem/article/55/4/611/5631762
Hi Bhaskar Chatterjee ,
You should use qRT-PCR, and along with the cancer cell, the normal cell also should be used. In this way, you can compare the regulation of the gene expression between cancer and normal cells (through comparison of Ct values of interest with a normal, which are both then normalized to a housekeeping gene). Please refer to https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036716/
Good luck
Hi,
To study the expression of any gene, be it upregulation or downregulation, you would need to study it's transcript (mRNA) and therefore abundance of it. Briefly, you will have to isolate Total RNA, separate mRNA from it, make cDNA, then perform Quantitative Real time PCR. You should have an experimental control and a Internal standard control (generally ribosomal RNA) and run them in parallel.
Quantitative RT-PCR data obtained can then be used to make comparisons.
Hello Bhaskar Chatterjee
In order to study gene expression by PCR you need to perform relative quantification by RT-qPCR. First you need to determine your target primer sequences whether or not these primers bind to exon-exon junctions otherwise you might end amplifying genomic DNA. Selection of candidate reference gene/genes for normalization is very crucial. They should have stable expression levels in your control as well as your experimental group. Various methods are available for calculation of relative gene expression like the Livak method, Pfaffl method depending on your assumption of your PCR efficiency. Please go through the MIQE guidelines which will give you a better understanding.
https://academic.oup.com/clinchem/article/55/4/611/5631762
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