Abi prism 7900 system

Total RNA was isolated by incubating the cells in a 25-cm² culture flask with 1 mL TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. After genomic DNA was removed by treatment with gDNA Eraser, RNAs were reverse-transcribed with random hexamer primers using PrimeScript RT Enzyme Mix I (PrimeScriptTM RT reagent Kit with gDNA Eraser, TaKaRa, Shiga, Japan). Real-time PCR were processed in triplicate and finished in 20 μL reaction volumes with SYBR^® Premix Ex Taq II (Tli RNaseH Plus, TaKaRa, Shiga, Japan) and the ABI PRISM^®7900 system (ABI). The threshold cycles and relative fold differences were calculated with 2^-ΔΔCt. The primers used in the study are listed in Table S1 and proved to be effective in previous studies (44 (link), 45 (link)).

RNA was isolated from cultured CFs using TRIzol (Thermo Fisher Scientific, Shanghai, China). Total RNA (0.5 μg) was reverse transcribed to cDNA. Real-time polymerase chain reactions were performed on an ABI Prism 7900 system. TaqMan probes to detect apelin, APJ, collagen I, collagen III, TGF-β, and α-SMA were purchased from Roche. All samples were amplified in triplicates for 45 cycles in a 384-well plate. The relative level of mRNA expression was expressed as 2^−ΔΔC_t. The primers are shown in Table 1.

Peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll-Hypaque (TBD, Tianjin, China) density gradient centrifugation from DM and SLE patients. The total RNA extracted from PBMCs using the TRIzol reagent (Invitrogen, Waltham, MA, USA) was reverse transcribed into cDNA with the Superscript II Reverse Transcriptase Kit (Takara, Shiga, Japan). The transcription levels of type I IFN-inducible genes (LY6E, OAS1, Mx-1, IFIT1, and IFIT3) were measured using SYBR^® Premix Ex Taq™ II (Takara, Shiga, Japan) via an ABI Prism 7900 system. The primer sequences we have previously reported (13 (link)) are as follows: LY6E: 5′-CTTACGGTCCAACATCAGAC-3′, 5′-GCACACATCCCTACTGACAC-3′; OAS-1: 5′-GAAGGCAGCTCACGAAAC-3′, 5′-TTCTTAAAGCATGGGTAATTC-3′; Mx-1: 5′-GGGTAGCCACTGGACTGA-3′, 5′-AGGTGGAGCGATTCTGAG-3′; IFIT1: 5′-TCAAAGTCAGCAGCCAGTCTCA-3′, 5′-GCCTCCTTGGGTTCGTCTATAA-3′; IFIT3: 5′-AACTACGCCTGGGTCTACTATCACTT-3′, 5′-GCCCTTTCATTTCTTCCACAC-3′; and GAPDH: 5′-ATTGCCCTCAACGACCACTTTG-3′, 5′-TTGATGGTACATGAAAGGTGAGG-3′. The IFN score was calculated as the sum of the five normalized expression levels of the corresponding tar (14 (link)):
Gene_{(DM or SLE)} represents the relative expression of a specific gene in DM or SLE patients. The average value $(\overline{X})$ and the standard deviation (SD) of the expression level of each target gene in healthy donors (HD, n = 10) were also presented.

Biopsy samples were rapidly cryo-sectioned and the tissues of normal squamous epithelia, SIL and SCC were isolated under microscopy. Tissues of about 20 mg or adherence cells in a 25-cm² culture flask were incubated with 1 mL TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) for RNA isolation in accordance with the manufacturer's instructions. RNAs were treated with gDNA Eraser to remove the genomic DNA and reverse-transcribed with random hexamer primers using PrimeScript RT Enzyme Mix I (PrimeScript^TM RT reagent Kit with gDNA Eraser, TaKaRa, Shiga, Japan). Real-time PCR was performed in a 20 μL reaction volume with SYBR^® Premix Ex Taq II (Tli RNaseH Plus, TaKaRa, Shiga, Japan) and the ABI PRISM^®7900 system (ABI). Reactions were processed in triplicate, and the threshold cycles and relative fold differences were calculated with 2^-ΔΔCt.

To examine the CSF cytokines mRNA, the ABI PRISM 7900 System was applied (Applied Biosystems, Foster City, CA 94404, USA). As stated by the manufacturer's instructions, the mRNAs for IL-1β, IL-6, IL-8, and TNF-α expression were established, while 18S was designated as a standard control owing to its stable expression under the stimuli of SAH. Each sample was launched into a TaqMan Human Cytokine Card that enclosed probes and primers for specific targets. This procedure was performed via an affixed filling reservoir and a vacuum loading process via the ABI PRISM Card Filling Station.
Target mRNAs were standardized according to the reference gene (18S), and final data were expressed as a relative fold from the baseline. Comparative mRNA expression was set by the Livak and Schmittgen ΔCT method [40 (link)]. The results were analyzed if a 5-fold increase in the mRNA levels compared with the baseline to allow for data consistency.

Real-time RT-PCR was performed using the ABI Prism7900 System (Applied Biosystems - Life Technologies, Saint-Aubin, France) as described [10 (link)] with PCR primers for LZTS1 (Hs00232762_m1) (Applied Biosystems). The ∆∆CT method was used to quantify transcripts.