I realize the answer to this may be more cell-type specific. We use human MSCs as well as human/mouse chondrocytes. Just wondering if anyone has had luck with a specific normalizer that works well for MSCs / chondrocytes or a wide range of other cell types?
Hi Audrey
The answer is not easy. Depending on the cell type you can use several of them. I worked with primary cells, and for them U6 and the SNORD family are usually good. You can also give a try to some low-abundance tRNAs like lysine-tRNA or arginine-tRNA.
Best. Good luck
Paco
Hi Audrey
The answer is not easy. Depending on the cell type you can use several of them. I worked with primary cells, and for them U6 and the SNORD family are usually good. You can also give a try to some low-abundance tRNAs like lysine-tRNA or arginine-tRNA.
Best. Good luck
Paco
If it is cultured cells, U6 generally works well, especially if the RNA quality is not a problem. More fancy ways to determine good normalizers is to look up microRNA expression datasets and look for microRNAs that do not change or change the least between all the samples. We did this for human skin tumor samples and came up with miR-423 and miR-5701.
For me the best choice is use multiple housekeeping (HK). This is particularly easy if you can have access to nanofluidic platforms such as Biomark (Fluidigm). We use in our chips three housekeeping we found highly expressed in multiple human tissues (U6, RNU44 and RNU48). This makes quantification very robust and reliable.
If you do not want (or can) use multiple HK, my suggestion is to look at the expression values of microRNA in the sequencing database and choose something which is consistently expressed. If you have a mix of mouse/human cells you may also consider to spike in in each sample a synthetic miRNA not expressed in both species before microRNA extraction. After designing a specific probe, you can then use this control to normalize without the potential problem coming from cross-reactivity between the two species of single miRNA you may use as HK.
My students and collaborators particularly use U6 generic design and RNU6B, but I have notice that besides stable expression similar expression helps in dendritic cells we used let7a.
Hi Audrey!
I concur with the colleagues: It depends on the cell type. I use 5s for cervical keratinocytes and tumor cell lines. It shows very low variation throughout the samples and is pretty abundant. U6 may be the answer to compare different cell types.
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