Abi prism sds 7000 pcr instrument

Total RNA isolation and real-time RT-PCR analyses for SOCS mRNA expression were performed as previously described [27 ]. The forward and reverse primers employed were as follows: for SOCS1, 5′-TTT TTCGCCCTTAGCGTGA-3′ and 5′-AGCAGCTCGAAG AGGCAGTC-3′; for SOCS3, 5′-AAGGACGGAGACTTC GATTCG-3′ and 5′-AAACTTGCTGTGGGTGACCAT-3′; for CXCL8, 5′-GCTGGCTTATCTTCACCATCATG-3′ and 5′-TTATTTTTTTTCAGTTAATTAACAGATGCT ATCAT-3′; and for β-actin, 5′-CATCGAGCACGGCATC GTCA-3′ and 5′-TAGCACAGCCTGGATAGCAAC-3′. The sequences of the primers and internal probe for HBD-2 mRNA have been previously described [4 (link)]. Primers for CIS (Hs003203371), SOCS2 (Hs00919620), SOCS4 (Hs00328404), SOCS5 (Hs00367107), SOCS6 (Hs00377781), SOCS7 (Hs00389987), CXCL1 (Hs00236937), and HPRT1 (Hs01003267) were purchased by Applied Biosystems (Branchburg, NJ, USA). Fluorescence intensity was analyzed by the ABI PRISM SDS 7000 PCR Instrument (Applied Biosystems). The fold induction value for triplicate wells was presented as the mean ± S.D.

Total RNA from keratinocytes and endothelial cells was extracted using the TRIzol reagent (InVitrogen), whereas total RNA from human and mouse skin biopsies by RNeasy Lipid Tissue Kit (Qiagen, Chatsworth, CA, USA). mRNA was reverse-transcribed into complementary DNA and analyzed by real-time PCR. GAPDH or β-2 microglobulin were used as housekeeping genes for human and murine mRNA, respectively. Primer pairs used in PCR reactions are listed in the table reported in Supplementary Table S1. Fluorescence intensity was analyzed by the ABI PRISM SDS 7000 PCR Instrument (Applied Biosystems, Branchburg, NJ, USA), using SYBR Green PCR reagents or Taqman PCR Master Mix. The values obtained from triplicate experiments were averaged, and data are presented as means ± SD.

Total RNA was extracted from keratinocyte cultures and human skin biopsies, by using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Lipid Tissue Kit (Qiagen, Chatsworth, CA, USA), respectively. mRNA was reverse-transcribed into complementary DNA by using SuperScript IV VILO reaction master mix (Invitrogen) and analysed by real-time PCR. GAPDH or β-actin were used as housekeeping genes, as specified in the Figure legends. Primer pairs used in PCR reactions are listed in the table reported in Supplementary Table 1. Fluorescence intensity was analysed by the ABI PRISM SDS 7000 PCR Instrument (Applied Biosystems, Branchburg, NJ, USA), using SYBR Green PCR reagents or Taqman PCR Master Mix. The values obtained from triplicate experiments were averaged, and data are presented as means of 2^-DDCT values ± SD.