Thymic epithelial cells were prepared as described previously (7 (link)) and then sorted using a FACS Aria II (BD Biosciences). RNA was isolated using Dynabeads® mRNA Purification Kit (Invitrogen) and reverse transcribed using SuperScript III Reverse Transcriptase and oligo d(T)16 primers (Invitrogen). qPCR was performed with TaqMan Gene Expression assays from Applied Biosystems (Gapdh, Mm99999915_g1; Aire, Mm00477461_m1). A Custom TaqMan assay (8 (link)) was used for mOVA transcripts.
For Copa transcript detection, RNA was prepared from the skin of indicated mice using RNeasy Mini kit (Qiagen). SuperScript III Reverse Transcriptase and oligo d(T)16 primers (Invitrogen) were used to synthesize cDNA. qPCR was performed with TaqMan Gene Expression assays from Applied Biosystems (Copa, Mm00550231_m1; Actb, 4352933E).
A Custom TaqMan assay was used for specific Copa transcripts: fwd: 5’-CAAGTGAAGATCTGGCGTATGAATG-3’, rev: 5’-AAAACAGCACAAGACACATTGTTGT-3’, probe for wildtype Copa allele: 5’-Vic-AGGCATGGGAGGTTGA-MGB-3’, probe for mutant Copa allele: 5’-Fam- AGGCATGGAAGGTTGA-MGB-3’.
Data were acquired with Bio-Rad CFX96 Real Time Machine.
For Copa transcript detection, RNA was prepared from the skin of indicated mice using RNeasy Mini kit (Qiagen). SuperScript III Reverse Transcriptase and oligo d(T)16 primers (Invitrogen) were used to synthesize cDNA. qPCR was performed with TaqMan Gene Expression assays from Applied Biosystems (Copa, Mm00550231_m1; Actb, 4352933E).
A Custom TaqMan assay was used for specific Copa transcripts: fwd: 5’-CAAGTGAAGATCTGGCGTATGAATG-3’, rev: 5’-AAAACAGCACAAGACACATTGTTGT-3’, probe for wildtype Copa allele: 5’-Vic-AGGCATGGGAGGTTGA-MGB-3’, probe for mutant Copa allele: 5’-Fam- AGGCATGGAAGGTTGA-MGB-3’.
Data were acquired with Bio-Rad CFX96 Real Time Machine.
