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Blood, Vol. 109, Issue 10, 4343-4350, May 15, 2007
GATA3 up-regulation associated with surface expression of CD294/CRTH2: a unique feature of human Th cells
Blood De Fanis et al.
109: 4343
Supplemental materials for: De Fanis et al, Vol 109, Issue 10, 4343-4350
Files in this Data Supplement:
- Table S1. Sequences of the primers synthesized for RT-PCR in this study (PDF, 67.6 KB) -
PCR conditions are available upon request. *Primers for PPIA generate a 320-bp amplicon with both human and mouse templates.
- Table S2. Transcript analysis in circulating CD4+CD45RO+ T cells sorted by CRTH2 expression (PDF, 38.8 KB) -
RNA was extracted and analyzed by quantitative RT-PCR as indicated in “Materials and methods.” Data are expressed relative to PPIA. Mean ± SEM of the indicated numbers of donors. Statistical significances are highlighted in red.
- Figure S1. Analysis and choice of primers for quantitation of human GATA-3 RNA by real-time RT-PCR (PDF, 56.3 KB) -
(A) A short (S) or long (L) amplicon generated with either human GATA-3 primer combination listed in Table S1 was electrophoresed on a 2% agarose gel. Shown is the electrophoretic mobility of reactions using either no template (N) or cDNA generated from in vitro–skewed Th1 (1) or Th2 (2) cultures or circulating CRTH2− (-) or CRTH2+ (+) memory Th cells from the same individual or a GATA-3–encoding plasmid as positive control (P; OriGene Technologies). (B) A melting curve was generated for either primer pair on an Applied Biosystems 7300 Real-Time PCR System. PD indicates primer dimer. The S primers were used throughout the study. A similar GATA-3 expression profile was observed in experiments using the L primers (panel A and not shown). As shown in panel A, an additional amplicon was generated in PCR reactions using these primers, suggesting detection of an alternatively spliced form of the gene. However, the biphasic melting profile shown in panel B (right) was not apparently due to this second amplicon, because a similar profile was observed with both cell-derived and synthetic templates (not shown).
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