Full-length gene sequences were extracted from WormBase (Release WS170) and primers were designed by the Primer3 software [31 ] and tested for specifity using NCBI BLAST. The targets amplified by the primer pairs were evaluated with MFOLD software [32 ] in order to check for the formation of secondary structures at the site of primer binding. MFOLD analysis was performed using default settings and 50 mM Na+, 3 mM Mg2+ and a temperature of 60°C (which is the annealing temperature of the primers). Primers were purchased from Invitrogen. Primer and amplicon information are listed in Table 3 .
Quantitative RT-PCR was carried out using a Rotor-Gene 2000 centrifugal real-time cycler (Corbett Research) using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Each reaction contained: 12.5 μl of the Platinum SYBR Green qPCR SuperMix-UDG, 200 nM, 300 nM or 400 nM of forward and reverse primers and 5 μl cDNA (1:40 RNA dilution), to a final volume of 25 μl. Amplification was performed in 0.1 ml real-time PCR tubes (Corbett Research) placed in the 72-well rotor of the Rotor-Gene instrument. The cycling conditions were as follows: 50°C for 2 min, initial denaturation at 95°C for 2 min, followed by 45 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C (gain set at 8 for SYBR Green). Following the final cycle, melting curve analysis was performed to examine the specificity in each reaction tube (absence of primer dimers and other nonspecific products). The Rotor-Gene software allows automatic melting curve analysis for all tested samples in a given run. SYBR Green fluorescence of the generated products was continuously monitored throughout the temperature ramp from 60 to 99°C. The temperature rose in 1° increments with a 5 s hold at each degree. A single melt peak for each reaction confirmed the identity of each PCR product. Each assay included a no-template control for every primer pair. In addition, aliquots of each reaction mixture were analyzed by agarose gel electrophoresis to evaluate amplification of nonspecific products.
Quantitative RT-PCR was carried out using a Rotor-Gene 2000 centrifugal real-time cycler (Corbett Research) using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Each reaction contained: 12.5 μl of the Platinum SYBR Green qPCR SuperMix-UDG, 200 nM, 300 nM or 400 nM of forward and reverse primers and 5 μl cDNA (1:40 RNA dilution), to a final volume of 25 μl. Amplification was performed in 0.1 ml real-time PCR tubes (Corbett Research) placed in the 72-well rotor of the Rotor-Gene instrument. The cycling conditions were as follows: 50°C for 2 min, initial denaturation at 95°C for 2 min, followed by 45 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C (gain set at 8 for SYBR Green). Following the final cycle, melting curve analysis was performed to examine the specificity in each reaction tube (absence of primer dimers and other nonspecific products). The Rotor-Gene software allows automatic melting curve analysis for all tested samples in a given run. SYBR Green fluorescence of the generated products was continuously monitored throughout the temperature ramp from 60 to 99°C. The temperature rose in 1° increments with a 5 s hold at each degree. A single melt peak for each reaction confirmed the identity of each PCR product. Each assay included a no-template control for every primer pair. In addition, aliquots of each reaction mixture were analyzed by agarose gel electrophoresis to evaluate amplification of nonspecific products.
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