Universal cdna standards

Total RNA was extracted using TRIzol reagent (Invitrogen, 15596–026). The cDNA synthesis was performed using PrimeScript RT reagent kit with gDNA Eraser (Takara, RR047A). Q-PCR experiments were conducted using SYBR Green Premix Ex Taq II kit (Takara, RR820A) and RT-PCR System-Applied Bio-system. The relative amount of mRNA expression of target genes was calculated by the comparative Ct method using GAPDH as a control. All Q-PCR reactions were performed in triplicate. Data was acquired using ABI ViiATM 7 Real-Time PCR System instrument. All primers for the 72 TRIM genes were validated using universal cDNA standards (BD Clontech). Quantification was performed by ΔCt method, with 18S or actin used for normalization. mRNA with cycle times ≥ 34 were determined to be undetected. Normalized mRNA levels are expressed as arbitrary units by transformed the cycle times using 2^ΔCt. The data were opposed to log2 and organized in a heat map using MEV software (Dana-Farber Cancer Institute, http://www.tm4.org/mev.html).

RNA isolation was performed as described previously38 (link). RNA was quantified using a NanoDrop 2000 (Nano-drop Technologies, Wilmington, DE, USA). The cDNA was synthesized with 2 μg of total RNA using an RT kit purchased from Thermo Scientific (USA). Real time PCR was performed using an SYBR green PCR master mix purchased from Roche (USA) and PCR-specific amplification was conducted in the Applied Biosystems ViiA^TM real-time PCR machine (ABI, CA, USA). All primers for the 72 TRIM genes were validated using universal cDNA standards (BD Clontech). The primer sequences for all TRIM genes were summarized in Supplementary Tables 1 and 2. Quantification was performed by the deltaCT method and GAPDH was used for normalization. Normalized mRNA levels were expressed as arbitrary units by transformed the cycle times using 2^−ΔΔCt (see Supplementary Tables 3 and 4). Hierarchical clustering analysis was performed on the normalized, log-transformed and median-centered RNA levels by calculating Pearson correlation as distance followed by average linkage analysis using Cluster2.11 software. The resulting cluster analysis was then displayed as a tree using TreeView1.60 software.