Atlas rt pcr primer sequences

Total RNA was isolated from liver or placenta using Ribopure (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Total RNA was subjected to DNase I treatment using Turbo DNA-free (Invitrogen, ThermoFisher Scientific, USA), and RNA integrity was confirmed by agarose gel electrophoresis. Afterwards, cDNA was synthesized by oligo(dT)-primed reverse transcription with Superscript II (Invitrogen, ThermoFisher Scientific, USA). qPCRs were performed using a Light Cycler 1.5 (Roche, Mannheim, Germany). The reaction solution was carried out in a volume of 20 μL, containing 10 pmol of both forward and reverse primers, 10× SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan) and the appropriate nanograms of the cDNA stock. Rps29 was used as a reference gene for qPCR. The primer sequences were obtained either from the Atlas RT-PCR Primer Sequences (Clontech, Palo Alto, CA, USA) or designed using the Primer3 software (University of Massachusetts Medical School, Worcester, MA, USA) [33 (link)]. Samples were analyzed in duplicate on each assay. Amplification of non-specific targets was discarded using the melting curve analysis method for each amplicon. qPCR efficiency and linearity were assessed by optimization of the standard curves for each target. The transcription was quantified by the Light Cycler Software 4.05 (Roche, Germany) using the efficiency correction method [34 (link)].