There is a set of primers that will amplify over the CDR3 region of the heavy chain Ig rearrangement. The 5' primer binds at the end of the IGHV region which is highly conserved between different IGHV regions, the 3' primer binds in the IGHJ region. They were designed for lymphoma/leaukaemia diagnostics. Of course they will not be perfect, even if they were they would be subject to interference by somatic hypermutation, but they are pretty good. I'm not sure about using them for qPCR, we last used them for B cell spectratyping with a semi-nest at the 3' end - so the primer details will be in Kate's paper (Aging Cell. 2009 Feb;8(1):18-25). Might be worth a try with the internal primers.

Hello,

In immunoproliferative disease oor acute lymphoblastic leukemia, there is still one rearrgement which is more present.

If you want to look at polyclonal cells, qPCR isn't possible because qPCR primers are designed for a specific Ig rearrangement.

So the best is to use cell immunophenotyping and flow cytometry to differenciatre mature b cells from non B cells or B cells precursors.

Targeting the rearranged V(D)J would indeed be a nice marker for mature B cells. However, it is problematic. One thing is that you will need primers that amplify all possible V and J genes, leaving you with a quite significant mix of primers in the PCR reactions. Additionally, these primers will bind both rearranged and non-rearranged V and J genes creating a quite messy PCR (would be difficult to design primers specifically to the V-D, D-J, or V-J junctions due to the junctional diversity). Theoretically though, you should only get successful amplification (PCR product of ~400 bp depending on where the primers are) of the recombined V(D)J as the non-recombined V and J genes are separated by thousands of bp in the genome.

I doubt this would be clean enough for a qPCR though. Maybe if you run a gel and see a specific band ~400 included in the other probable mess of side products.

There is a set of primers that will amplify over the CDR3 region of the heavy chain Ig rearrangement. The 5' primer binds at the end of the IGHV region which is highly conserved between different IGHV regions, the 3' primer binds in the IGHJ region. They were designed for lymphoma/leaukaemia diagnostics. Of course they will not be perfect, even if they were they would be subject to interference by somatic hypermutation, but they are pretty good. I'm not sure about using them for qPCR, we last used them for B cell spectratyping with a semi-nest at the 3' end - so the primer details will be in Kate's paper (Aging Cell. 2009 Feb;8(1):18-25). Might be worth a try with the internal primers.

Thanks to all of you! The assay proposed by Deborah looks most promising as it employs only 3 primers even though generating a spectrum of products they fit perfectly to qPCR range.

I am agree with Marck Muller and Johannes Truck, even I have worked with B 40 system in NIHT3 cells. I have never used PCR to clear the difference between both kind of subsets. It will be easy with flow citometry, But if you are looking for another specific result in B cells , then use PCR or qPCR

Looking at VDJ rearrangements would not distinguish a mature B cell from a pre-B cell, which has already undergone HC gene rearrangement, either partial DJ or complete VDJ depending on which definition of a pre-B cell you are using.

Yes Peter and Johannes are right. I didn't miss those words but I did read it differently. If you want to distinguish all B cells including preB cells from others then my original answer stands. If you want to distinguish all B cells except preB cells then you need a different method. Using IgD constant region primers would identify all mature B cells that were not class switched, but you would miss immature B cells and class switched B cells/plasma cells. It really depends what you are looking for. All B cells after preB will have light chain rearrangements so you could go for those. I haven't used a set of light chain primers that span all light chain CDR3s. In theory it will be possible to design something but a bit messy - there is both kappa and lambda to account for and for each of these the consensus is not as clean as for heavy chain.