Hi all,
I currently have a puzzle when seeking to use PCR to compare some genes expression across different cell lines. I usually work within one single cell line. But I think the logic is okay as if ddct value shows A cell line got higher expression of a gene than B cell line, then we can safely conclude the this gene got higher expression in A cell line. Is it a fair comparison?
Any suggestion or comment?
This might be of some interest to you. Not as an answer to your question, but as a source of ideas to consider.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for confirming gene expression differences or measuring transcript abundance due to its sensitivity, reproducibility, simplicity and high-throughput. With this assay, the common method is normalization of gene expression using an endogenous reference gene. Ideal reference genes should be sufficiently abundant and have stable expression across different tissues and cell lines under different experimental conditions, but the ideal and universal reference gene does not exist in practice. Inaccurate normalization can cause inadequate quantification and incorrect conclusions. Currently, several mathematical approaches including geNorm, NormFinder, Bestkeeper and comparative delta Ct have been developed to assist appropriate reference gene selection and geNorm provides a measure of the minimum optimal number of reference genes to normalize.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305315/
Hi Zhuoran,
Yes, we can compare the expression profiles by the use of PCR provided you have a suitable marker gene and good primers targeting the marker gene, along with an appropriate control. And by PCR I assume you are talking about the Quantitative real time PCR (qRT-PCR).
By RTPCR, it is fair comparison, you can further confirm by immunohistochemistry, or in situ hybridization or even northern blotting. It depends how much resources you want to put in. But RTPCR is easy step,
Thanks all. From what you all have suggested, it seems that it is feasible to use rt-PCR to conduct the gene expression comparison across various cell types. Our lab currently has GADPH and beta-actin as housekeeping genes. Hopefully from that it could produce meaningful results.
By the way, any reference article I could learn from? I might need to cite their work just in case.
Hello.
The choice of GAPDH and ACTB as reference genes for analysis of relative levels of mRNA is inadequate because they have pseudogenes and their expression is unstable. I strongly recommend you to read several reviews regarding the problem of choosing correct reference genes:
1) Bartłomiej Kozera & Marcin Rapacz. Reference genes in real-time PCR. J Appl Genetics (2013) 54:391–406. DOI 10.1007/s13353-013-0173-x
2) Chapman JR, Waldenström J (2015) With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS ONE 10(11): e0141853. doi:10.1371/journal.pone.0141853
3) Lieven Thorrez et al. Tissue-specific disallowance of housekeeping genes: The other face of cell differentiation. Genome Res. 2011. 21: 95-105. doi: 10.1101/gr.109173.110
Hello
Yes you can and this link will help you
Article Identification of Valid Reference Genes for the Normalizatio...
Good Luck
yes you can use a semiquantitative RT-PCR and then run the PCR products on the agarose gel to detect the gene bands and upon thier intensity you can compare their expression.
This might be of some interest to you. Not as an answer to your question, but as a source of ideas to consider.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for confirming gene expression differences or measuring transcript abundance due to its sensitivity, reproducibility, simplicity and high-throughput. With this assay, the common method is normalization of gene expression using an endogenous reference gene. Ideal reference genes should be sufficiently abundant and have stable expression across different tissues and cell lines under different experimental conditions, but the ideal and universal reference gene does not exist in practice. Inaccurate normalization can cause inadequate quantification and incorrect conclusions. Currently, several mathematical approaches including geNorm, NormFinder, Bestkeeper and comparative delta Ct have been developed to assist appropriate reference gene selection and geNorm provides a measure of the minimum optimal number of reference genes to normalize.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305315/
yeah if you are ding Real time pcr then you can
http://www.gene-quantification.de/
Thank goes to Mohammed and Elena. I start to understand the tricky part on choosing a correct Housekeeping gene for PCR assay. I will check your recommendations and may further inquire you for more information!
Thank also goes to others for your kind help.
From what I understand, PCR can compare gene expression across different cell types, but to do this I need to find out the most proper HK genes to work with. If I can use the known antibody to test out the post-transnational product in the protein level via western blot, then is it more convenient because by then I can get around this HK gene issue?
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