For SYBR green quantitative reverse-transcriptase PCR (qRT-PCR), cDNA was transcribed from 0.5 µg of total RNA using Superscript III (Invitrogen). SYBR green reagents and huCK2α, huCK2α′ and (human glyceraldehyde 3-phosphate dehydrogenase) huGAPDH normalization primers were obtained from SABiociences, and used as described (25 (link)).
For endpoint PCR, total RNA was isolated from spleens using TRIzol Reagent (Invitrogen). Total RNA (0.5 µg) was used as template to perform RT-PCR using Titan One Tube™ RT-PCR system (Roche Molecular Diagnostics). Primers used were: 5’-GTTGCTTCCCGATACTTCAAAGG-3’ (CK2α’, F), 5’-GAACCTTGGCTATCCTCACCAAC-3’ (CK2α’, R), 171 bp product; 5’-TCCTCGTGGACTATCAGATG-3’(CK2α’, F), 5’-AACCTTGGCAATGCGAAC-3’ (CK2α’, R) 140 bp product; 5’-CCCTTCATTGACCTCAAC-3’ (GAPDH, F) 5’-TTCACACCCATCACAAAC-3’ (GAPDH, R); 120 bp product. The PCR amplification included RT 50°C for 30 min, 94°°C for 4 min, 33 cycles of 94°°C, 45 sec; 55°C, 20 sec; 72°C, 45 sec with final 5 min extension at 72°C. For normalization, parallel RT-PCR reactions were performed with GAPDH primers using the same conditions. PCR products were separated by 1.0% agarose gel and stained with ethidium bromide. Images were acquired using a Kodak digital imaging system and quantitated using ImageJ.
For endpoint PCR, total RNA was isolated from spleens using TRIzol Reagent (Invitrogen). Total RNA (0.5 µg) was used as template to perform RT-PCR using Titan One Tube™ RT-PCR system (Roche Molecular Diagnostics). Primers used were: 5’-GTTGCTTCCCGATACTTCAAAGG-3’ (CK2α’, F), 5’-GAACCTTGGCTATCCTCACCAAC-3’ (CK2α’, R), 171 bp product; 5’-TCCTCGTGGACTATCAGATG-3’(CK2α’, F), 5’-AACCTTGGCAATGCGAAC-3’ (CK2α’, R) 140 bp product; 5’-CCCTTCATTGACCTCAAC-3’ (GAPDH, F) 5’-TTCACACCCATCACAAAC-3’ (GAPDH, R); 120 bp product. The PCR amplification included RT 50°C for 30 min, 94°°C for 4 min, 33 cycles of 94°°C, 45 sec; 55°C, 20 sec; 72°C, 45 sec with final 5 min extension at 72°C. For normalization, parallel RT-PCR reactions were performed with GAPDH primers using the same conditions. PCR products were separated by 1.0% agarose gel and stained with ethidium bromide. Images were acquired using a Kodak digital imaging system and quantitated using ImageJ.
