Viiatm7 instrument

Total RNA was isolated using Trizol (Invitrogen Corp, San Diego, CA, USA). The first strand cDNA was synthetised using the M-MLV reverse transcriptase (Promega Corporate, Wisconsin, USA). Quantitative real time polymerase chain reaction was performed in duplicates using the SYBR Premix Ex Taq polymerase (Takara Bio Inc.) on a ViiA^TM 7 Instrument (Applied Biosystems). Results were obtained after 40 cycles of a thermal step protocol consisting of 95 °C (1 s), 60 °C (20 s). The sequences of primers were reported in Supplemental Table S3. Gene expression data were normalized to the expression levels of 18S (ΔCT values). Means of ΔCT or of ΔΔCT values (versus untreated cells) were calculated and results were represented as 2^−ΔCT or 2^−ΔΔCT. Statistics were performed on ΔCT or on ΔΔCT individual values.

Isolated RNAs were reverse transcribed into the first strand cDNA by using SuperScript III (Thermo Fisher Scientific). The transcribed cDNAs were diluted into an appropriate concentration and used as templates to perform PCR reactions with Hieff ® qPCR SYBR Green Master Mix (Low Rox) (Yeasen). These reaction systems were then analyzed on the ViiATM7 instrument (Applied Biosystems) according to the instruction. To ensure the accuracy of results, TUB 8 acts as an internal control and each independent sample contains at least three biological replicates and two technical replicates. All used primers are listed in Additional file 6: Table S5.

Melanocyte RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) followed by purification with the PureLink RNA Mini Kit (Life Technologies, Grand Island, NY), which includes a DNAse digestion step. Since melanin has been reported to interfere with PCR [29 (link)], RNA samples were purified using the OneStep™ PCR Inhibitor Removal Kit (Zymo Research Corporation, Irvine, CA), and cDNA was synthesized using an Omniscript RT kit (Qiagen, Valencia, CA).
Real time PCR was performed using a ViiA^TM7 instrument (Applied Biosystems, Foster City, CA) with the Quantitect SYBR green PCR kit (Qiagen, Valencia, CA). Primers (Table 1) were designed in our laboratory or based on previously published sequences [30 (link)]. A dissociation curve for each primer set ensured amplification of a single specific product. The comparative C_t method (2^-ΔΔCt) was used to quantify gene expression levels (), where ΔΔC_t = ΔC_t(sample)– ΔC_t. For all results, the sample represents melanocytes cultured with dermal fibroblast-conditioned medium and the reference represents control melanocytes cultured with an empty insert. Sample and reference were normalized to the endogenous housekeeping gene, GAPDH; dermal fibroblasts had no significant effect on melanocyte GAPDH cycle thresholds. Data are reported as mRNA fold change.

Total nucleic acid was extracted from frozen stool using the QiaAmp stool DNA kit (Qiagen, Valencia, California) and used in the Enteric Pathogen TaqMan^® Array Card (TAC) as previously described [23 (link)]. Briefly, 40 μL of extracted nucleic acid from each stool sample was mixed with 60 μL of Ag-Path-ID One-Step RT-PCR kit (Applied Biosystems, Foster City, CA) and this mixture was loaded onto the eight ports of the TAC, sealed, and loaded into the ViiATM7 instrument (Applied Biosystems). TAC can detect the following pathogens: bacteria: Aeromonas, Bacteroides fragilis, Campylobacter (C. jejuni and C. coli), Clostridium difficile, EAEC, EPEC, ETEC, Helicobacter pylori, Salmonella, Shigella/EIEC, STEC, and Vibrio cholera, fungi: Encephalitozoon intestinalis and Enterocytozoon bieneusi, nematodes: Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis, and Trichuris trichiura, protozoan parasites: Cryptosporidium, Cyclospora, Entameoba histolytica, Giardia A/B, and Isospora and viruses: adenovirus, astrovirus, norovirus GI/GII, rotavirus, and sapovirus [23 (link)]. Analysis of raw data files were processed using ViiATM7 software version 1.2.2 (Applied Biosystems) as previously described [23 (link)]. A threshold cycle (Ct) greater than 35 was used as the analytical cutoff (lower limit of detection).