FIG. 1.

Optimization of input template mRNA to avoid distortion of CDR3 length distribution resulting from template insufficiency. (A) CDR3 length distributions distorted by insufficient template were characterized as a warble baseline, low FI, and missing length peak of 201 bp in Vβ13 and a complete loss of length peaks in Vβ17. (B) Amplification of Vβ2 using serially diluted template mRNA at concentrations ranging from 10 ng (from 25.0 × 10⁴ purified T cells) to 0.6 ng (from 1.6 × 10⁴ purified T cells). The minimum amount of template mRNA able to generate a Gaussian distribution of CDR3 lengths was 1.3 ng. Two long CDR3 length peaks (**) diminished at the 0.6-ng level. (C) TCR CDR3 within CD45RA and CD45RO CD8 T cells from a healthy adult was amplified for 18 Vβ families by using 1.3 ng mRNA template. All Vβ families were amplified without a baseline oscillation and with the FI within the normal range, including Vβ families showing clonal expansion (Vβ15 within CD8 CD45RA T cells and Vβ11, Vβ14, Vβ15, and Vβ18 within CD8 CD45RO T cells). (D) Representative TCR CDR3 length distribution of all 21 Vβ families within CD45RA and CD45RO CD4 T cells from a 15-year-old healthy subject with the amount of template (4 to 20 ng mRNA) used for this study. The CDR3 lengths of all Vβ families showed Gaussian distributions.