housekeeping genes for brain tissue - (Sep/29/2008 )

Hi,
are there any housekeeping genes specially suited for brain tissue apart from beta-actin and GAPDH? We perform RT-PCR from hippocampal feline tissue.
Greetings, Barbara

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Selection of the right housekeeping gene depends on different factors and is dependent on the kind of material AND experimental circumstances you are working with. the ideal HKG should
-not be up-/downregulated in your experimental setup
-be in the same concentration (Ct / CP) range (+/- 6 Cts)
-expressed in all samples
-well designed assay (efficiency, specificity, sensitivity)

some examples for HKGs:
G6PD Gene Assay (glucose-6-phosphate-1-dehydrogenase)
GAPD Gene Assay (glyceraldehyde-3-phophate dehydrogenase)
GUSB Gene Assay (β-glucuronidase)
ACTB Gene Assay (β-actin)
PGK1 Gene Assay (phosphoglyceratekinase 1)
HPRT Gene Assay (hypoxanthine-guanidine phosphoribosyltransferase)
PBGD Gene Assay (porphobilinogen deaminase)
β2M Gene Assay (β2 microglobulin)
TBP Gene Assay (TATAA-box-binding protein)
PPIA Gene Assay (peptidylprolyl isomerase A / Cyclophilin A)

Always try to avoid 18-S as it is overrepresentaded in all samples and can easily inhibit your target gene in multiplexing experiments.

Sometimes it is even a good strategy to use the averaged ratio to 2 HKG instead of using only 1 HKG.

Which assay format (Sybr Green / TaqMan Probes) and evaluation-software / algorithm / instrument are you using?

Good success!

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Hi, Steph,

many thanks for your reply. I am afraid not to be able to answer your questions exactly, therefore I describe our experimental design. Our aim is to investigate cell cycle markers in feline neurons of hippocampus (cyclins and cyclin-dependent kinases). I do cryosections, stain the sections and afterwards we perform laser-capture-microdissection (we simply cut out the neurons under microscopical obeservation and try to avoid any other cell type represented in the brain). We isolate RNA from the cells and do Real-Time-PCR (we used GAPDH and beta-actin as hk-genes). The problem is that cells in the brain usually contain less RNA, even the hk-genes are at 28-30 cycles in RT-PCR.
As I am not performing RT-PCR by myself, I am not experienced with this method.

Furthermore I have real basic questions concerning the definition of the term "housekeeping gene" - 1) are the quantities of housekeeping gene-expression different in different cell types and 2) is the quantity of housekeeping gene-expression different between cells of the same cell type dependant on the localisation (e.g. is housekeeping gene-expression in neurons of the hippocampus different from that of neurons in other brain areas?)

Greetings from Vienna,
Barbara

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I'd avoid beta actin. It can be regulated in brain tissue after stimulation.
You do cyclines, tho, so I don't know really...

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