Rnaquous 4 pcr kit

Embryos were injected at the one- to two-cell stage with 25–50 pg of circular etsrp-XeX as previously described (Sumanas et al., 2008 (link)) in addition to scl MO. Batches of 20 injected and control uninjected embryos were frozen on dry ice at the tail bud stage. Total RNA was purified using the RNAquous-4PCR kit (Ambion). cDNA was synthesized using Superscript III reverse transcriptase and oligo-dT primer (Invitrogen). Real-time PCR was performed using Chromo4 thermal cycler (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). The following PCR profile was used: 95°C 5 min; 95°C 1 min, 58°C 1 min, 72°C 1 min, detection at 82°C for 10 sec; steps 2 through 5 repeated 44 times. Relative cDNA amounts for mpx were calculated using the Opticon 3 software (Bio-Rad) and normalized to the expression of elongation factor 1 α (EF1α). Primers for measuring mpx expression span an intron-exon boundary to detect cDNA instead of genomic DNA. The following primer sequences were used. mpx-Forward: CTG CGG GAC CTT ACT AAT GAT GG; mpx-Reverse: CCT GGA TAT GGT CCA AGG TGT C; EF1α-Forward: TCA CCC TGG GAG TGA AAC AGC; EF1α-Reverse: ACT TGC AGG CGA TGT GAG CAG