I'm getting trained on performing B cell PCR; however, I understand that DNA-RNA-protein and if our goal is to produce the antibody, then it may make sense to amplify RNA directly, but do we have to? Would it be possible to perform it from DNA of the Ig from sorted B cells? Would having the DNA reduce cost and also it being more stable?
Lastly, has anyone try to perform B cell PCR on certain number of B cells instead of the whole sorted 96-well plate as normally? I am just trying to see if we could save some money on this strategy, but afraid of the final yield.
Thank you very much in advance.
Hi
I believe the most scientist using RT-PCR on the RNA for evaluation of gene expression of Ig. You cannot use the DNA for this purposes. You will just determine presence of Ig genes in the DNA, not more. If you will need evaluate the level of expression in a different conditions you must use the RT-qPCR.
Hi Quang,
As you know, reverse transcription PCR amplifies the mRNA available within the cells. Whilst a B cell might have your gene of interest within its DNA, it is possible that the gene isn't actually transcribed, and therefore translated into proteins. Just because the cell has the DNA of your desired protein, doesn't mean it is actually being produced by the cell, and mRNA at least confirms that the gene is being transcribed thus closer to confirming the gene of interest is being produced.
Hope that helps!
I see. That makes sense. I was just trying to reduce the cost associated with steps in the whole process. Thanks for your answers.
Hi,
you can amplify DNA of sorted B cells but it is difficult to set up a proper PCR because of aspecific annealing of the primers throughout the whole genome. Working with cDNA produced with specific primers for Ig gene (reverse primers in the constant region) is much easier as you will amplify only the Ig variable region. Anyway amplifying Ig fragments from sorted polyclonal B cells won't give you much information because you will obtained overlapped sequences of the many different specificities of the B cells. This approach only works if you then go for deep sequencing, otherwise you have to work at monoclonal level.
You can find some information in
Best wishes,
Nat Med. 2004 Aug;10(8):871-5. Epub 2004 Jul 11.
An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.
Traggiai E1, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A.
Actually it is possible to amplify Ig genes(I mean rearranged V-(D)-J genes) from genomic DNA. In this case you have to use primers at the downstream end of the J region, or in the adjacent non coding region. The advantage to do PCR on RNA is that you can use primers in the C region that is shared with all Ig of the same classe. This more simple in some instance.
The problem you may face is the clonality of your B cells, since if you have several kind of B cells with each own rearrangements, then sorting or cloning is necessary.
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