Expression of Antimicrobial Peptides: Materials and methods

16 Jan
2011

Subjects

Punch skin biopsies were taken from involved and adjacent uninvolved skin of 7 patients with AD after TEWL measuring. AD was diagnosed according to the standard criteria(Hanifin and Rjaka, 1982). Normal skin was obtained from healthy donor. Informed consent was obtained from all subjects after they were fully informed about the details and the potential risk of the study, and the study was ap­proved by the Ethical Committee of Chung-Ang University Hospital Institutes Review Board.

TEWL assessment

All subjects condition were first stabilized for 15 to 20 minutes in a climate and humidity-controlled room. Ambient temperatures ranged between 21° and 24° , with a mean relative humidity of 45%. TEWL was measured with a Tewameter TM 210 (Courage & Khazaka, Cologne, Germany) and estimated over representative involved skin sites as well as adjacent clinically normal appearing un­involved skin with AD. TEWL levels were also measured over normal skin of healthy donor.

Preparation of primer

We synthesized the PCR primer from the basis of Gene Bank data. Primers were chemically syn­thesized by using DNA synthesizer (Pharmacia, Bjo :gatan, Uppsala, Sweden). Their sequences were as follows:

hBD-2 (128bp):
5-ATC ТСС TCT TCT CGT ТСС TC-3′ (sense), 5′-ACC TTCTAG GGC AAA AGA CT-3 (anti-sense) LL-37 (208bp):
5-CTG ATG CCT CTT CCA GGT GT-3′ (sense), 5′-GAG GGA GCC CTT TCT GAA TC-3′ (anti-sense) GAPDH (593bp):
5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′ (sense), 5′-GGT GCT GCT TGT TAG GAG GTC AAG TAA AGG GC-3′ (anti-sense)

Reverse transcription-polymerase chain reaction
(RT-PCR)

The tissues were cut (hash) by scissor. Total RNA was isolated from skin using TRIZol reagent (Invitrogen, Carlsbad, CA, USA) after adding 1 ml of TRIZol reagent. And the tissue homogenized by homogenizer. After 5 minutes at room temperature, 0.2 ml of chloroform per 1 ml of TRIZol reagent was added. Tubes were shaken vigorously by hands for 15 seconds and incubated at 15° to 30° for 3 minutes. The mixtures were centrifuged with 12,000 rpm at 4° for 15 minutes, the upper aqueous phase were transferred to a fresh tube, and the same amount of 2-propanol was added. After mixtures were incubated at 4° for 15 minutes, it was centrifuged with 12,000 rpm at 4° for 15 minutes. The supernatant was removed, then washed 500 ul of 70% ethanol with 12,000 rpm at 4° for 5 minutes, the RNA pellet was briefly dried. The purified RNA was dissolved in DEPC-DW 30 yl. Three yg of total cellular RNA was reverse transcribed at 42° for 30 minutes in a 20 yl volume contating 1 yl reverse transcriptase (TaKaRa, Shiga, Japan), 10 x >uffer 2 yl, lOmM dNTP 2 yl (dNTP mix), oligo dT primer 1 yl, RNase inhibitor 0.5 yl, 25 mM MgCl2 4 yl 2 yl of each cDNA sample from the RT-PCR was amplified by PGR in 25 yl containing 10 x >uffer 2.5 yl, 25 mM MgCl2 2.5 yl and 10 pmol 0.75 yl primer.
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Thermal cycle profiles were as follows; 94° for 5 minutes, 35 cylces of 94° for 1 minutes, 59° for 1 minutes, 72° for 1 minutes, final extension step of 72° for 10 minutes. RT-PCR analysis of LL-37 and hBD-2 mRNA using specific primers were performed.

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