Abi prism sds software

Total RNAs were isolated from 2 × 10⁵ cells using TRIzol reagent (Invitrogen) and MaXtract High Density (Qiagen). First-strand cDNA was synthesized by using the random hexamer primers in the SuperScript III system (Invitrogen). Quantitative real-time TR-PCR (qRT-PCR) was performed using the Applied Biosystems ABI Prism SDS software and the following primers: for ISG15, 5ʹ-GCTGGGACCTGACGGTG-3ʹ (sense) and 5ʹ-TTAGCTCCGCCCGCCAG-3ʹ (anti-sense); for UBE1L, 5ʹ-AGGTGGCCAAGAACTTGGTT-3ʹ (sense) and 5ʹ-CACCACCTGGAAGTCCAACA-3ʹ (anti-sense); for UbcH8, 5ʹ-AACCTGTCCAGCGATGATGC-3ʹ (sense) and 5ʹ-TGGTGCAAGGCTTCCAGTTC-3ʹ (anti-sense); for Herc5, 5ʹ-GGGATGAAAGTGCTGAGGAG-3ʹ (sense) and 5ʹ-CATTTTCTGAAGCGTCCACA-3ʹ (anti-sense); for β-actin, 5ʹ-AGCGGGAAATCGTGCGTG-3ʹ (sense) and 5ʹ-CAGGGTACATGGTGGTGCC-3ʹ (anti-sense).

Nematodes were synchronized and harvested at L4 stage. RNA and cDNA were prepared as described (22 (link)). For semi-quantitative RT-PCR, the PCR cycles were as follows: fat-6, 22 cycles; pod-2 and cdc-42, 25 cycles; fat-5 and fat-7, 28 cycles; elo-2, 30 cycles. Real-time PCR assays were run and monitored with an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). The real-time PCR was conducted on at least three treatment groups with each individual treatment group at least in triplicate. Threshold values (Ct) for the gene of interest and a reference gene, cdc-2, were determined using ABI Prism SDS software version 1.1 (Applied Biosystems). The expression level of the gene of interest was evaluated using the 2^−(ΔΔCt) method (23 (link)). Supplementary Table 4 shows primer sequences used for the quantitative real-time RT-PCR.

Total mRNA was extracted from 1 × 10⁶ cells by Manual PerfectPure^TM RNA Cell & Tissue (5PRIME, GmbH). RNA was reverse transcribed using the high-capacity cDNA Archive Kit (Applied Biosystems, Foster City, USA) and real time PCR were performed as previously described [21 (link)]. Finally, raw data were analyzed by the ABI prism SDS software (Applied Biosystems) and primers (Ct1) mRNA expression data were obtained from Ct values, normalized to 18 s RNA (Ct2) (housekeeping) content and finally expressed as 2^-Δct for each gene.

Total RNAs were isolated from 2 x10⁵ cells using TRI reagent (Molecular Research Center, Inc) and a MaXtract High Density tube (QIAGEN). cDNAs were synthesized using random hexamer/oligo d(T) primers provided by the QuantiTect Reverse Transcriptase Kit (QIAGEN). Quantitative real-time RT-PCR was performed using SYBR green PCR core reagents (Applied Biosystems) and ABI Prism SDS software. PCR was performed using the following primers: 5′-GACATCCCTGAGATTAAG-3′ (for IFNβ forward), 5′-ATGTTCCTGGAGCATCTCG-3′ (for IFNβ reverse), 5′-GCTTAGACATATTCTGAGCCTAC-3′ (for CXCL10 forward), 5′-AGCTGATTTCCTGACCATCATTG-3′ (for CXCL10 reverse), 5′-AGAAATCAAGGGAGAAAGAA-3′ (for ISG54 forward), 5′-AAGGTGACTAAGCAAATGGT-3′ (for ISG54 reverse), 5′-AGCGGGAAATCGTGCGTG-3′ (for β-actin forward), 5′-CAGGGTACATGGTGGTGCC-3′ (for β-actin reverse), 5’-ACTTTTTCCTGGCTCATCTC-3’ (for PKR forward), 5’-ACATGCCTGTAATCCAGCTA-3’ (for PKR reverse), 5’-GCTGGGACCTGACGGTG-3’ (for ISG15 forward), 5’-TTAGCTCCGCCCGCCAG-3’ (for ISG15 reverse), 5’-CAGAAAGCAGTGCGGCCCCT-3’ (for USP18 forward), 5’-TGCAGGGCGTCCTCCAGTGT-3’ (for USP18 reverse), 5’-CACACCAGCGTTTTGACCAG-3’ (for ChREBP forward), 5’-ACTCAAACAGAGGCCGGATG-3’ (for ChREBP reverse), 5’-CAACCTGCTGAAGGAGAAG-3’ (for c-Fos forward), and 5’-AGATCAAGGGAAGCCACAGAC-3’ (for c-Fos reverse).