Moloney murine leukemia virus

Based on the manufacturer's instructions, Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue samples. The ratio of OD260: OD280 was 1.8-2.0 in all samples. The integrity of RNA was detected by agarose gel electrophoresis. Based on the manufacturer's guidelines, Moloney murine leukemia virus (Promega, Madison, WI, USA) was used to reverse transcribe RNA samples into complementary DNA.
The SYBR® Premix Ex Taq (Tli RNaseH plus) qPCR kit (TaKaRa Biotechnology, Inc., Shiga, Japan) and ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) were used for qPCR detection. The primers are listed in Table 2. As mentioned earlier, the amplification efficiency of each primer was determined by using the double continuous dilution of cDNA (32 (link)). The amplification efficiency of GAPDH was nearly 100%. The magnification outcomes were confirmed by two methods: agarose gel electrophoresis and sequencing. As mentioned earlier, the relative expression of target gene mRNA was analyzed by the 2^−ΔΔCt method (33 (link)). Gene expression was normalized to GAPDH and presented as relative fold change compared to the NC group. All samples were analyzed in triplicate.

Plates of FACS-sorted cells were thawed and a combined lysis/denaturation step was performed by incubation at 70°C/4 min, 4°C/5 min, 2.5 µl reverse transcription master mix was then added to each well [1 µl 5× RT buffer (Promega), 0.6 µl 35 mM MgCl₂, 0.25 µl Moloney murine leukemia virus (Promega), 0.1 µl 25 mM dNTPs, 0.5 µl H₂O] and incubated at 37°C/2 min, 42°C/1 min, 50°C/1 s for 40 cycles, then 85°C/5 min, and 4°C hold. Following RT, cDNA was preamplified by adding 2 µl of cDNA from the RT plate to 8 µl of preamp master mix [5 µl TaKaRa premix Taq polymerase (Clontech), 2.5 µl 0.25X TaqMan pool, 0.5 µl H₂O] and thermocycled at 95°C/3 min, 55°C/2 min, 72°C/2 min, then 95°C/15 s, 60°C/2 min, 72°C/2 min for 16 cycles, and 4°C hold. Amplified cDNA was then diluted 1:50 in nuclease-free H₂O and this material was used for qPCR against a curated panel of 48 TaqMan Gene Expression Assays (Table 1) on 48.48 dynamic arrays using a Biomark HD system (Fluidigm).

To validate transcriptome results, DCs obtained from patients with T1D (n ≥ 4) and control subjects (n ≥ 3) were cultured and pelleted in three conditions: iDCs, PSAB-DCs and mDCs. RNA was isolated using RNeasy Micro Kit (QIAGEN), and was reverse-transcribed with a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). cDNA synthesis reactions were carried out using random hexamers (0.5 mg/ml, BioTools, Valle de Tobalina, Madrid, Spain) and reverse transcriptase Moloney-murine-Leukemia-virus (200 U/ml, Promega, Madison, WI, USA). Quantitative RT-PCR assays were performed with TaqMan universal assay (ThermoFisher Scientific) on a LightCycler^® 480 (Roche, Mannheim, Germany) using the following TaqMan assays: CYTH4 (Hs01047905_m1), GIMAP4 (Hs01032964_m1), HPGD (Hs00960590_m1), NFKB inhibitor alpha (NFKBIA) (Hs00153283_m1), PLAUR (Hs00958880_m1), TNFAIP3 (Hs00234713_m1), tumor necrosis factor superfamily member 14 (TNFSF14) (Hs00542477_m1), and VEGFA (Hs00900055_m1). Relative quantification was performed by normalizing the expression for each gene of interest to that of the housekeeping gene GAPDH (Hs02758991_g1), as described in the 2^–ΔCt method (28 (link)).

Commercial RNA extraction kit (RareRNA, Bio-East Technology, Taipei, Taiwan) was used to isolate cellular RNA, as previously described [11 (link)]. The DNase I kit (Invitrogen, Carlsbad, CA, USA) was used to prepare DNA-free RNA solution. In the experiment, 5 µg of total RNA as starting material, 5 µg of oligo(dT)15 primer (Life Technologies, Carlsbad, CA, USA), and 200 units of reverse transcriptase (Moloney murine leukemia virus; Promega, Madison, WI, USA) were used to synthesize complementary DNA (cDNA) at 42 °C for 45 min. Real-time quantitative polymerase chain reaction (RT-qPCR) amplification was conducted using a standard TaqMan PCR protocol on ABI StepOnePlus system (Applied Biosystems, Foster City, CA, USA). The primers used for RT-qPCR are listed in Table 1. All samples were tested in duplicate wells. The ∆Ct (threshold cycle) was calculated by subtracting of the average glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ct values from the target genes Ct values, which reflects the target gene mRNA level. The change of CBS, CSE, and 3-MST gene expression were calculated as 2^−∆Ct, and the fold change of genes was presented compared to control group.