Abi sds v2

Reverse transcription, performed on 1 μg of TRIzol (Sigma-Aldrich, St. Louis, MO)-extracted total RNA, was followed by cDNA synthesis using the AMV reverse transcriptase kit (Roche, Indianapolis, IN). qPCR was performed with the help of TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA), using primers and 6-carboxyfluorescein (6-FAM) / Black Hole (BHQ)-labelled probes to specific target genes (IDT, Coralville, IA). Sequences of probes are available upon request. The Applied Biosystems 7900 HT device was used for amplification and detection of PCR product. ABI SDS v 2.3 software (Applied Biosystems, Carlsbad, CA) was employed for analyzing results. The Ct value for each gene was normalized to the internal reference control, HPRT1 for human genes or Hprt for mouse genes, and represented as fold gene expression change relative to gene expression in normal human or mouse B cells or, in case of drug studies, to vehicle-treated cells.

Total RNA was extracted using RNA-IsoPlus (Takara) and cDNA was synthesized by PrimeScript RT Master Mix (Takara). qRT-PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems) and primers as listed in Supplementary Table 1 on an ABI Prism 7900 System with data analyzed using the ABI SDS v2.3 software (Applied Biosystems). Relative expression differences were calculated using the 2^−ΔΔCt method.

Cells were resuspended and lysed in Trizol (Invitrogen) and complementary DNA (cDNA) was synthesized using a reverse transcription-PCR Kit (Roche) according to the manufacturer's instructions. qRT-PCR was performed using SYBR Green PCR Kit (Applied Biosystems) on a 384-well plate with an ABI PRISM 7900 Sequence Detector (Applied Biosystems). The results were analyzed using the ABI SDS v2.4 software (Applied Biosystems). The sequences of primers used are listed in Table S1.

Total RNA was extracted using TRIZOL Reagent (Invitrogen), and cDNA was synthesized using a reverse transcription (RT)-PCR Kit (Roche) according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green PCR Kit (Applied Biosystems) and an ABI PRISM 7900 Sequence Detector (Applied Biosystems). Specificity of primers was verified by dissociation curve analysis. Data was analyzed using ABI SDS v2.4 software (Applied Biosystems). All qRT-PCR reactions were performed in duplicates. Housekeeping gene GAPDH was used as an internal control. Using primers listed in Supplementary Table 5.