Fig. 3.

Blood cells transcribe self-antigen genes, including a proinsulin RNA splice variant. (a) RNA was freshly isolated from PBMC, reverse-transcribed into cDNA with random hexamer oligonucleotides, and amplified by PCR using nested sets of sequence-specific primers that spanned introns to avoid amplifying genomic DNA. GAPDH was used as a housekeeping gene. Electrophoresis in 1% agarose and staining with ethidium bromide demonstrated cDNAs for proinsulin, GAD65, and 21-OH in all five individuals examined (one example shown). Product identities were confirmed by DNA extraction and sequencing. (b) Native (N) and splice variant (SV) proinsulin RNAs from the pancreas and flow-sorted PBMC were quantified by real-time PCR (Upper), and the PCR products were identified by staining with ethidium bromide after electrophoresis in 1% agarose (Lower).