I was trying to analyse expression levels of certain genes after treatment of monocytic THP1 cell line with antigenic proteins through qPCR and i tried using the reference genes beta actin and GAPDH but for both of them Cq values are not same for treated and untreated cells. Is there any way that i know the reference genes that are stable under the treatment? or should it depend on the cell line also? that case what about using macrophage cell lines?
Unfortunately it is difficult to tell you a good reference gene for your conditions. You should experimetnally determine it. The best is to pick up several putative reference genes for your condition taken either from literature or from other sources and check them by qPCR. Then, do a box plot with the Cts and pick up the one(s) that have best behaviour when compared under your conditions (do not change). If taking several use an average of the Cts for referencing.
@Miguel López de Heredia sir can you please tell me more about getting putative reference genes
There are two way to find the best reference gene that suits your needs:
1. Design/order primers for several reference gene and run with your treated/untreated samples. (I would suggest to start with ß2m, 18s and HPRT)
2. Buy a commercial pcr array like http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-000A.html and test your samples.
I do not know this cell line but I have found the paper in the link. Perhaps it helps you for selecting other genes for testing. You could also try the ones Korcan has suggested you. What we normally do is try 4 or 5 genes and then take the bests. This is different each time and we look for the genes according to the cell lines and tissues we are working with. Hope it helps. As mentioned, you will have to look for the genes that does not change in your conditions and that will be a kind of trial and error until you get them. If you have data from a microarray from those experiments, you could always look into the data for genes that do not change in your conditions.
http://www.biomedcentral.com/1471-2199/11/90
I have worked with the line THP 1 and usually use as the ACTB which shows Cq of about 17-18 in sybr green RT-PCR with 50 ng of cDNA.
Normally its advisable to use more then one reference gene. For THP-1 in most cases RPL13 and HPRT(1) are usefull.
Its always advisable to use more then one reference gene for normalization in qPCR. I would use 18S rRNA (most stable) and HPRT. Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line