Expression of the 24 zinc transporter genes in non-stimulated peripheral blood mononuclear cells. Data are shown as boxplots representing 25, 75 percentile, median and 5–95 range. The means are represented by the small square. The skewed distribution, particularly for the 5 genes with very low expression levels, is due to the presence of samples without any gene expression (see text ‘Expression of zinc transporters’). Note: all ‘negative samples’ cannot be illustrated, due to the logarithmic scale of the y-axis. A.u., arbitrary units; Zn2+, zinc.
Expression of the 24 zinc transporter genes in stimulated peripheral blood mononuclear cells (PBMCs). The expression levels for the 24 zinc transporter genes were determined in stimulated PBMCs. These data were normalized to the level of the corresponding non-stimulated cells. The horizontal line illustrates factor 1 that showed no change between stimulated and non-stimulated PBMCs. Due to the variability among the 5 samples, data are shown as a histogram. The small circles illustrate individual experiments. Increased and decreased expression are represented by values >1 and <1, respectively. The small lines represent the median of the dataset. The samples of proband no. 2, which exhibited a different expression pattern, are represented by the gray circles. Note: all ‘artificial values’ (for details see ‘Data presentation, calculation of transcript levels and statistics’ for ZIP2, ZIP5, ZIP12, ZnT3 and ZnT10) are marked by a cross within the circle. Due to the similar extent of upregulation (e.g., ZIP14, 1.27-fold, 1.28-fold) all the ratios are not illustrated. A.u., arbitrary units; Zn2+, zinc.
Primer sets for qRT-PCR.
| Gene/accession number | Name | Sequence of primers | Size of the PCR product (bp) |
|---|---|---|---|
| ZnT1 | F: 5′-CCAACACCCTGGTGGCCAATAC-3′ | 212 | |
| R: 5′-TGAACCCAAGGCATCTCCAAGG-3′ | |||
| ZnT2 | F: 5′-CTGCGCTGTGGCTGTGAACATC-3′ | 345 | |
| R: 5′-TCCACCGACAGCAGCAGATCAC-3′ | |||
| ZnT3 | F: 5′-CACTGGCATCCTCCTGTACCTG-3′ | 332 | |
| R: 5′-GGTCGGCTGCCTTGTATTGAG-3′ | |||
| ZnT4 | F: 5′-GCGCCATCATACTCACCCTG-3′ | 275 | |
| R: 5′-GTGACGGTGACCAGACTGGTTC-3′ | |||
| ZnT5 | F: 5′-TTAAACATGCAGTTGCTGGGTG-3′ | 329 | |
| R: 5′-ACCCTTGTGATCTGCCACACC-3′ | |||
| ZnT6 | F: 5′-TTTGGAACAGCCCGAGATACAC-3′ | 260 | |
| R: 5′-AAAGAGCAAATGCTCCAGCAAG-3′ | |||
| ZnT7 | F: 5′-GGAAGTTCTGGCTGGCTTTGTC-3′ | 278 | |
| R: 5′-GGCTATGGCAATGATCGACATG-3′ | |||
| ZnT8 | F: 5′-TGCCTTTCAGCGAATTCACAAC-3′ | 311 | |
| R: 5′-GTGCTGCATGTGTCAGAGGGTC-3′ | |||
| ZnT9 | F: 5′-AAATACCAAGCCACGCTCCAG-3′ | 200 | |
| R: 5′-TGCCCAATGCTAGTAAACCCTG-3′ | |||
| ZnT10 | F: 5′-GACCGTGTTCGCAAACGTAGC-3′ | 282 | |
| R: 5′-TTGATAAGCGGGAAGGCAGATG-3′ | |||
| ZIP1 | F: 5′-CGGCCAGGAGCTAACCATGAAG-3′ | 307 | |
| R: 5′-GCCCACCATTCACTGTTCCCAG-3′ | |||
| ZIP2 | F: 5′-ACTCTGGGCTGTGGCCTTACTC-3′ | 284 | |
| R: 5′-AAGCCCAGGGAGATGATGAGC-3′ | |||
| ZIP3 | F: 5′-TGGTTGGGCTCGGTAGCACATC-3′ | 68 | |
| R: 5′-GTCACTGCAGGGCCAAACCATC-3′ | |||
| ZIP4 | F: 5′-CAACAGCTCCAGTGTGTGGGAC-3′ | 319 | |
| R: 5′-GCTCAGGAAGGTCTGCAGGATG-3′ | |||
| ZIP5 | F: 5′-TCATTGGCTGACCACCTGAATG-3′ | 293 | |
| R: 5′-AGCAAGGGCCGTAGTAGACGAG-3′ | |||
| ZIP6 | F: 5′-CATGGCATGGGCATCCAGGTTC-3′ | 289 | |
| R: 5′-TCAAAGTCCCAACGGCCAGTGC-3′ | |||
| ZIP7 | F: 5′-CAGGACCTGGATGCTGTCACTC-3′ | 319 | |
| R: 5′-CGACAAGAAAGGCAACAATTCC-3′ | |||
| ZIP8 | F: 5′-CTGCCATCAATGGTGTGACATG-3′ | 253 | |
| R: 5′-CCCTGAAGGAGAGACAAGGTGC-3′ | |||
| ZIP9 | F: 5′-TGCTGGCCTTCTCTGTGGAAC-3′ | 302 | |
| R: 5′-TGCTAGACCTTGCTGCTTCTGG-3′ | |||
| ZIP10 | F: 5′-GCATAATCGGGTCCACAAACC-3′ | 318 | |
| R: 5′-AACAATGCAGGGCAAAGGTATG-3′ | |||
| ZIP11 | F: 5′-CGGCATCTGCTACCTTTGAGAG-3′ | 269 | |
| R: 5′-ATGATGTCGTCCATGACCACG-3′ | |||
| ZIP12 | F: 5′-CGAATACCCTCCGCCTATCAG-3′ | 299 | |
| R: 5′-TGCTGGATGATCCCTGGACTC-3′ | |||
| ZIP13 | F: 5′-CTCTTGGGCAATGTGTTTCTGC-3′ | 300 | |
| R: 5′-GACTTTGATGCTCCGGACCAC-3′ | |||
| ZIP14 | F: 5′-CTCTGTGTGACCGTCATCTCCC-3′ | 315 | |
| R: 5′-AAGCGACTCAGAGGCATAATGG-3′ |
Standard protocol: initial step at 95°C for 15 min; 40 cycles at 94°C for 30 sec, at 57°C for 30 sec and at 72°C for 30 sec; and annealing at 72°C for 5 min. qRT-PCR, quantitative reverse transcription-polymerase chain reaction.
Qualitative change of zinc transporter gene expression patterns of individual samples.
| PBMC sample no. | No. of induced zinc transporter genes (ratio >1, Fig. 3) | No. of reduced zinc transporter genes (ratio <1, Fig. 3) |
|---|---|---|
| 1 | 4 | 20 |
| 2 | 15 | 9 |
| 3 | 2 | 22 |
| 4 | 4 | 20 |
| 5 | 7 | 17 |
Absolute numbers represent the numbers of transporter genes either induced (ratio induced/control cells >1) or reduced (corresponding ratio <1) for the 24 genes. PBMC, peripheral blood mononuclear cell.