Well - interesting question. If B2M is mutated such that it won't express or otherwise be available, then Class-I won't be on the surface regardless of how much mRNA there is. So that's one thing.

Yes Class-I is very polymorphic, but only in particular regions of the molecule. While I'm not an expert, I'd be surprised if you could not find primers that would allow you to PCR from (say) "constant regions" of HLA-A, HLA-B, HLA-C and quantitate them without regard to the specific histological allele.

As with all qRT-PCR, pay close attention to controls, don't chose one that is a single exon, and no matter how you get rid of gDNA run a check using a non-expressed unique sequence such as a promoter (we used to run CD4 promoter). Do NOT fall into the trap of saying you don't need to check for gDNA because your primers "cross an intron".

Good Luck!

G

Thank you, sir. I did try to find the conserve region between HLA-A,B and C (outside of exon 2 and 3 since both of the exon code for alpha1 and alpha2 which is the polymorphic region of the peptide groove). However, the conserved regions (HLA-I is still too diverse) seems to be too short (not continuous) for primer design (qPCR) and the primers designed is unavailable to target all the histological allele (this explains why researchers prefer WB and IHC due to mAb and pAB (created via hybridoma technique) that can targets HLA-A, B and C). Due to the compound phenotype of altered HLA Class I in cancer, I am now trying to decide which is the best HLA type to target. A cancer causing mutation is affecting the HLA at many levels (transcriptional, PTM, protein etc) so mRNA detection HLA might give inconclusive results depends on the mutation types and stages of the cell lines. The only way left is for me to study the whole antigen processing machinery, the related molecules (TAP, LMP etc), all HLA Class I regulatory elements (which might be affected in cancer cells) just to ensure that the changes is in the mRNA level. I utilized COSMIC for extensive search for mutations in the cancer cell lines genes (HLA-I and their regulatory elements, or regulator of the regulator) but it seems like finding a needle in a haystack. Even multiple research provide discordance in level of specific HLA (A, B, C etc) expression in metastatic tumours. However, thank you for your advice, I really appreciate it. Have a good day, sir.

Hi again

forgive me if I wasn't clear, I meant a constant region for HLA-A, and a different one for HLA-B and so on, not a "universal Class-I" one.

Besides - while HLA is polymorphic in the population, each individual has a maximum od 2xA, 2xB, 2xC. So your MCF7 cells won't really be polymorphic at all....

This may be useful:

Article A catalog of HLA type, HLA expression, and neo-epitope candi...

Good Luck

G

Dear Ahmad Hafiz Murtadha,

In my eyes it is not possible, to detect changes in MHC Class I expression using qPCR in MCF7 with one primer pair, only.

There are a lot of HLA-class-I and HLA-class-I-like genes and pseudogenes.

To give some examples: A, B, C, E, F, G, H, MICA, C, D, E, F ... !

And all, or at least the most, would react in your qPCR with your primer pair.

Please have a closer look to: 'Gene map of the extended human MHC' (nature Reviews Genetics, 2004).

Best regards,

Thomas

Reference:

https://www.nature.com/articles/nrg1489

'Gene map of the extended human MHC' (nature Reviews Genetics, 2004)