Rnaqueous 4pcr

Total RNA was prepared using RNAqueous-4PCR (Ambion, Life Technologies, St Aubin, France). Reverse-transcription was performed with high-capacity cDNA reverse-transcription kits (Applied Biosystems, Life Technologies). Resulting cDNA was amplified in triplicates by the SYBR-Green PCR assay. PCR reactions were incubated for 2 min at 50 °C and for 10 min at 95 °C, followed by 40 amplification cycles with 1 min annealing/extension at 60 °C and 15-s denaturation at 95 °C. Quantitative real-time PCR of mouse CCL19 and CCL21 was performed by the comparative threshold cycle (ΔΔC_T) method and normalized to mouse HPRT1 using AB 7,900 HT real-time PCR system (Applied Biosystems). The primer sequences used for CCL19 identify functional CCL19 (ref. 50 (link)). Primer sequences were as described50 (link): CCL19 forward: 5′-CTGCCTCAGATTATCTCGCAT-3′, CCL19 reverse: 5′-GTCTTCCGCATCATTAGCAC-3′; CCL21 forward: 5′-ATCCCGGCAATCCTGTTCTC-3′, CCL21 reverse: 5′-GGTTCTGCACCCAGCCTTC-3′; HPRT1 forward: 5′-CCTTCACCAATGACTCCTATGAC-3′, HPRT1 reverse: 5′-CAAGTTTACAGCCAAGATTCAC-3′.

The iQ SYBR Green supermix and iScript cDNA synthesis kits were purchased from Bio-Rad (Hercules, CA). The RNAqueous-4PCR and DNA removal kits were obtained from Ambion (Austin, TX). The avidin-biotin reaction (ABC) kit was ordered from Vector Laboratories (Burlingham, CA). Other chemicals were purchased from Sigma (St. Louis, MO).

Two to three weeks after virus injection, the cerebellar flocculi of two DKO mice injected with L7::H2-D^b-T2A-copGFP virus were dissected for mRNA analysis by RT-PCR. Thalamus from one WT control and spleen from one DKO mouse were used as positive and negative control samples, respectively. Primers for H2-D^b were designed to detect exon 2 and exon 3 regions of H2-D^b. RNA was extracted from each sample using RNAqueous-4PCR (Ambion, Life Technologies, NY) and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). PCR products were evaluated by gel electrophoresis to confirm the presence of PCR products of predicted size of ~250 bp. H2-D^b primers:
Sense- 5’CAAGAGCAGTGGTTCCGAGTGAG-3’;
Antisense- 5’CTTGTAATGCTCTGCAGCACCACT-3’.
Reactions for RT-PCR were carried out as previously described (Lee et al., 2014 (link)) using 1 ug of cDNA as a template (5 min at 95°C followed by 40 cycles (30 s at 95°C, 30 s, at 60°C, 30 s, 72°C)) (Veriti 96-well Thermal Cycler, Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. GAPDH primers: Sense- 5’ATTGTCAGCAATGCATCCTGC-3’ Antisense- 5’AGACAACCTGGTCCTCAGTGT-3’. The quality of cDNAs was confirmed by genotyping PCR reactions as described previously (Lee et al., 2014 (link)) using 0.5 ~ 1 µg of cDNA as template and the samples containing genomic DNAs were discarded.