I am wondering if anyone who is well versed with VDJ sequencing for TCR repertoire analysis (specifically CDR3) would know if DNA or RNA is a better starting material? We are looking at the effects of radiation therapy on immune system of mice. Thanks

  • $\begingroup$ DNA is typically easier to work with. Just make sure the cells you're working with have actually matured enough and undergone VDJ recombination. $\endgroup$
    – MattDMo
    Jan 29 '21 at 2:14
  • $\begingroup$ I agree with @MattDMo Colleagues next door have done this with single sorted cells which where sequenced afterwards. $\endgroup$
    – Chris
    Jan 29 '21 at 7:15
  • $\begingroup$ @MattDMo @Chris♦ how do you suggest one makes sure T cells have matured and undergone VDJ recombination?? I have looked at literature and seen one study that looked at clonal expansion 22 days after radiation exposure but they did not say why. Also, I have read that RNA is better to use than DNA since DNA won’t give you the antigen specificity you can see with mRNA expression. I have also seen that DNA can only show you the possible combinations of VDJ you could get as mRNA shows you the ones you did get, in real time. $\endgroup$ Jan 29 '21 at 18:12
  • 1
    $\begingroup$ I suggest you spend some time looking at the details of somatic recombination. Early during maturation, the V, (D,) J, and C protogenes recombine in the DNA to form the first version of the recombined TCR $\alpha/\gamma$ and $\beta/\delta$ chains (different protogenes combine in different ways in the different chains). These recombined sequences are then transcribed as mRNA - to my knowledge the protogenes are never transcribed. So, mRNA sequences should match DNA sequences. $\endgroup$
    – MattDMo
    Jan 30 '21 at 22:59
  • 1
    $\begingroup$ The reason I gave my caveat is because if you just take a whole thymus and do single-cell sequencing, you'll also get immature T cells that haven't undergone recombination. Instead, I would use either blood-derived cells, or sort thymocytes using a marker that is expressed after recombination. I would choose blood, because some of the recombined thymocytes will die during further maturation, and probably shouldn't be included in your measurements of TCR diversity. $\endgroup$
    – MattDMo
    Jan 30 '21 at 23:01

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.