i'm working on a substitution variant in HIST3H3 gene on the K122 residue associated with breast cancer. acetilation of this residue intensifies the transcription of down-stream genes. this residue in the mutated form, coudn't be acetylated.
pictures show the expression pattern of two different internal controls (GAPDH1 and Beta-actine) in response to overexpressoion of mutated/normal genes.
i used constant amounts of cDNA in all experiments.
do my ICs work correctly?
I would like to see the graph for raw reads in both the cases of your internal control. Check with n=3 replicates. If the raw reads are equal in all the experimental condition, go for it. Otherwise you need to check other internal controls such as, 18SrRNA or 28SrRNA. Thanks.
I would not be surprised if GAPDH or ActB changed expression in response to something potentially global like a histone mutation. Frankly, GAPDH and ActB are often extremely poor reference genes, changing expression under a lot of conditions.
People seem to use them as reference genes for no better reason than "they work well at the protein level, so they must at the mRNA level", or worse "because everyone else is using them".
I would recommend you take a representative panel of samples (ideally ~10 or more, with equal representation from your mutant and WT) and run qPCRs with a variety of candidate genes: then take the results from that and analyse them using geNorm, bestkeeper or normfinder (or all three). These algorithms empirically determine the most stable genes under your experimental conditions, and if all three are in rough agreement, this is strong evidence the selected genes are good references.
Here's the list of human genes from the primerdesign geNorm kit (for an example of a candidate panel). Note GAPDH, ActB and 18S are included (primerdesign always include these genes, even if they're unstable, purely so you can empirically determine that they are unstable).
Human geNorm kit gene list
Homo sapiens actin, beta (ACTB), mRNA. Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNA. Homo sapiens ubiquitin C (UBC), mRNA. Homo sapiens beta-2-microglobulin (B2M), mRNA. Homo sapiens phospholipase A2 (YWHAZ), mRNA. Homo sapiens Ribosomal protein L13A (RPL13A), mRNA. Homo sapiens 18S rRNA gene Homo sapiens cytochrome c-1 (CYC1), mRNA. Homo sapiens eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), mRNA. Homo sapiens succinate dehydrogenase complex, (SDHA), mRNA. Homo sapiens topoisomerase (DNA) I (TOP1), mRNA. Homo sapiens ATP synthase, (ATP5B), mRNA
Thanks, Dr. Hildyard!
do you think there is an HKG which its expression doesn't change in response to histone's mutations at all?
Isn't this better to choose another method, such as absolute expression, for my intention?
does this alteration in IC's expression confirm the carcinogenicity of the mutation of interest?
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