Hi everyon!
It's a long question, and I hope I'll be able to explain myself well.
I did a RT-PCR reaction for CD11b with a housekeeping genes panel, to pick one that best suited my cells.
Overall, for all the HK genes I used, there was a rapid elevation (Ct values of 18-21), and their melting curves looked good too, one uniform peak. However, I came across two phenomena that I am not sure how to relate to, and how they affect the selection of the best HKG for me:
1. In the melting curve plot, most of the genes present a peak at around the same temperature (80oC), apart from two exceptions. One presents its peak at a higher temperature (87oC), and another at a lower temperature (77oC).
Is there any preference for choosing a HKG based on these results?
Moreover, does the height of the peak matters in some way (the value according to the-Y axis)?
2. My experiments are conducted on microglia cells (WT vs. Mut.), and I use neurons as a control.
Some of the HKG I checked got the same Ct for the 2 types of microglia, but with a difference of 1-2 cycles from the neurons. Others, got the same Ct values for the neurons and only one of the microglia types, while the other type of elvated in an interval of about one cycle, for the same HKG.
What do you think is preferable, that my HKG would be as uniform as it can be for my experimental cells (microglia) or should I try to take one that would fit best for all of the cell types? I thought the latter may be preferable, so I chose to use ActB, which was the most uniform among all cell types.
To ensure different HKG wouldn't lead to different conclusions, I normalized the results of the CD11b to each HKG. There were some diffrences, of course, but for ActB (that I planned to chose) I got a 2.5 fold diffrence from the other HKG.
I'm not sure what to do.. Do you have any recommendations?
There are several free programs for analysing housekeeping genes as potential reference genes. I have used GeNorm, BestKeeper, and Normfinder. They give different results so try at least two programs and compare. I test 6-12 genes for each experiment and the best genes differ each time. I won't go into detail because I look at age and regional variations in the brain, so cultured cells will have different expression patterns of housekeeping genes.
Melting temperature depends largely on size of amplicon and is not relevant to actual expression. Use it to check all reactions have amplified the correct product. Run a gel with a few wells for each target and your no-template control to make sure your Ct's are valid.
If your neurons are your control, then the housekeeping gene you choose must be stably expressed in all cell types.
Lastly, there is no perfect housekeeping gene to use a reference. You can only minimise the variation. Therefore, it is best to use 2-3 genes at least and use their geometric mean to normalise your target genes.
Hi @Jennifer Blackburn,
Thank you very much for the detailed answer!
I looked for the programs you've mentioned, but only NormFinder is for free. Do you know about other recommended algorithm\software? I tried to google it and found just one, another paid software..
Dear Nofar
Make sure you are aware of housekeeping gene pitfalls: I suggest you look into the article
Rocha-Martins M, Njaine B, Silveira MS. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development. PLoS One. 2012;7(8):e43028.
Best regards
Rafael Linden Dear Rafael,
Thanks for your comment and the article. I will read it thoroughly.
Hi Nofar. Bestkeeper is free from http://download.gene-quantification.info/ (there is a link to get a password) and a free version of GeNorm is available in R (http://bioconductor.org/packages/release/bioc/html/SLqPCR.html) described in https://www.gene-quantification.de/vandesompeles-2002.pdf.
Sorry for the late reply!
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