Viitm7 real time pcr system

Total RNAs were extracted using TRIZOL Reagent (Invitrogen). In each reaction 500 ng of total RNA was used to generate cDNA. RNA samples were mixed with DNase (Invitrogen, CA, USA) to get rid of unwanted genomic DNA and incubated with PrimeScript RT Master Mix (Takara). The mixture was incubated at 37 °C, 15 min; 85 °C, 5 s. The cDNA was diluted in 150 µl DNase/RNase-free water and stored at −20 °C until use. Aliquots (3 µl each) of cDNA were amplified using Power SYBR Green PCR Master Mix (Takara) and ViiTM7 Real-Time PCR System (Applied Biosystems). Each reaction was performed in triplicate and GAPDH was used as an internal control. The primers are listed in Supplementary Table 3.

Total RNA was isolated using TRIzol (Invitrogen). 0.1 μg of total RNA was reverse-transcribed into cDNA with iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The resulting cDNAs served as the templates for Quantitative-PCR analysis using iTaq Universal SYBR Green Supermix (Bio-Rad) and ViiTM7 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). Primers for specific genes are: Mouse β-actin, 5′-TGACGTTGACATCCGTAAAGACC-3′ and 5′-AAGGGTGTAAAACGCAGCTCA-3′; Mouse IFNβ, 5′-CCCTATGGAGATGACGGAGA-3′ and 5′-CTGTCTGCTGGTGGAGTTCA-3′.

Total RNA was extracted from liver tissues stabilized in RNAlater. Extraction, cDNA synthesis and reverse transcription quantitative PCR (RT-qPCR) for vitellogenin Aa (vtgAa) transcripts were carried out, as described previously [37 (link)]. Elongation factor alpha (EF1α) was used as a housekeeping gene to normalize transcript abundance. All RT-qPCR was conducted on a Vii^TM 7 Real-Time PCR System (Applied Biosystems, Willmington, DE, USA).