Superscript 3 reverse transcriptase

Total RNA was isolated using RNeasy Kit (Qiagen, Valencia, CA, USA) from cells or heart tissue according to the protocol of the manufacturer, respectively. Reverse transcription to cDNA was performed with 30 ng of total RNA, random primers, and SuperScript III Reverse Transcriptase (Roche, USA). The qPCR was performed using a Light Cycler 480 SYBR Green I Master (Roche, USA) and the MiniOpticon qPCR System (Bio-Rad, CA, USA). After denaturation for 10 min at 95 °C, the reactions were subjected to 45 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. GAPDH was used as the internal standard control to normalize gene expression using the 2^-ΔΔCt method. The sequences of the qPCR primers were listed in Supplementary files 2 and 3.

RNA was isolated using the RNeasy minikit (Qiagen) or TRIzol followed by DNase I treatment (Invitrogen). RNA was reverse-transcribed with SuperScript III reverse transcriptase (Roche) and random hexamers, and qPCR was performed on a 7900 real-time PCR system (Applied Biosystems) using Power SYBR green (Applied Biosystems). Oligonucleotides are shown in Supplemental Table S8.

Total RNA was isolated from skin explants using Tri Reagent (Sigma) and treated with RQ1 DNAse (Promega) to remove contaminating genomic DNA. cDNA was synthesised from total RNA using random primers and Superscript III reverse transcriptase (Roche) in a 20-μl reaction. Reactions were diluted 20-fold and had 3 μl used as a template for each qRT-PCR. Each reaction was performed in a 20-μl volume using Universal SYBR Green Master Mix (Roche) containing Rox reference dye. Reactions were performed in triplicate, with at least 3 biological replicates used to determine each data point. Relative expression levels were determined from cDNA dilution standard curves and normalised to Tbp (or Capzb for half-life determination). Oligonucleotide sequences used are given in S1 Supporting Methods.

Cellular RNA was extracted using an RNeasy minikit (Invitrogen) following the manufacturer’s directions. cDNA corresponding to the viral mRNA of segment 6 (NA) was synthesized using tagged primers, thereby adding an 18-to-20-nucleotide tag at the 5′ end as follows: CCAGATCGTTCGAGTCGT. Reverse transcription was performed with the tagged primer as described by Lanford et al. (1994) (68 (link)). An 8-μl mixture containing the approximately 200 ng of total RNA and 10 pmol of tagged primer was heated for 10 min at 65°C, chilled immediately on ice for 5 min, and then heated again for 5 min at 60°C. A 12-μl volume of preheated reaction mixture (10 μl First Strand buffer [Invitrogen; 2×] and 2 μl Superscript III reverse transcriptase) was added and incubated for 1 h.
Quantitative PCR (qPCR) was performed with a SYBR green qPCR kit (Roche; catalog no. 507203180) using a LightCycler 480 system (Roche). A 3-μl volume of a 10-fold dilution of the cDNA was added to the qPCR reaction mixture (5 μl SYBR green qPCR mix, 1 μl of 2 μM forward primer, and 1 μl of 2 μM reverse primer). The qPCR cycles were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The primers used were as follows: mRNAtag (reverse), 5′-CCAGATCGTTCGAGTCGT-3′; WSNseg6_1314F (forward), 5′-TGAATAGTGATACTGTAGATTGGTCT-3′.

For in vivo experiments, the retina was collected from the eyes of EAU mice 19–21 days after immunization and were homogenized for RNA extraction. For in vitro experiments, naïve CD4⁺ T cells were cultured under Th17 differentiation conditions for 3 days. Total RNA was extracted from these samples using RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions, quantified, and then converted to cDNA using SuperScript III Reverse Transcriptase (Roche). qRT-PCR was performed using LightCycler 480 Sybr Green I Master (Roche) using LightCycler 480 II real-time PCR (Roche Applied Science). Gene expression was normalized to that of the housekeeping gene Gapdh and fold change was calculated by using the 2^−ΔΔCT threshold cycle method. Relative gene expression was normalized by comparing gene expression to the average of controls that was set to 1. A list of primers is presented in Supplementary Data 1.