For PCR, 4 μl cDNA was incubated with 30.5 μl water, 4 μl
25 mM MgCl2, 1 μl dNTP, 5 μl 10× PCR buffer,
and 0.5 μl (2.5 U) platinum Taq DNA polymerase (Gibco) and
the following primers (2.5 μl each containing 10 pmol):
bactericidal-permeability–increasing (BPI) protein (forward
primer: 5′-TTCCAGCTTCCCAGTTCCCAGATA-3′; reverse primer:
5′-CATCCACGGCAGGGTAGAAGGTAA-3′, 841 bp); CAP37,
heparin-binding protein (forward primer:
5′-AGCATGAGCGAGAATGGCTACGAC-3′; reverse primer:
5′-GGTCCTCGGGGGTCACAGTCA-3′, 235 bp), LL-37, human cationic
antimicrobial protein (forward primer: 5′-ATCATTGCCCAGGTCCTCAG-3′;
reverse primer: 5′-GTCCCCATACACCGCTTCAC-3′, 251 bp), HD5, humanα
-defensin 5 (forward primer: 5′-GCCATCCTTGCTGCCATTC-3′; reverse
primer: 5′-AGATTTCACACACCCCGGAGA-3′, 241 bp), HD6, human α-defensin 6
(forward primer: 5′-CCTCACCATCCTCACTGCTGTTC-3′; reverse primer:
5′-CCATGACAGTGCAGGTCCCATA-3′, 269 bp), HBD-1, human β-defensin 1
(forward primer: 5′-TTGTCTGAGATGGCCTCAGGTGGTAAC-3′; reverse primer:
5′-ATACTTCAAAAGCAATTTTCCTTTAT-3′, 253 bp), and HBD-2, humanβ
-defensin 2 (forward primer: 5′-CCAGCCATCAGCCATGAGGGT-3′; reverse
primer: 5′-GGAGCCCTTTCTGAATCCGCA-3′, 255 bp). Thirty-five cycles were
performed with each primer pair (annealing temperature 60°C). A
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific
intron-spanning primer pair (0.1 μM; 5′-CCAGCCGAGCCACATCGCTC-3′;
5′-ATGAGCCCCAGCCTTCTCCAT-3′), which yielded a 360-bp amplified product,
served as the internal control for equal amounts of cDNA. All primers
were synthesized by MWG Biotech AG, Ebersberg, Germany.
The positive control cDNA samples analyzed included one sample from
human blood (BPI, CAP37, LL-37), one sample from cultured epithelial
cells (HBD-1), and one sample from small intestine epithelium (HD5 and
HD6). The cDNA was replaced with water for a negative control reaction.