Rt2 qpcr primer assay 200

Chromogranin A gene (CHGA) expression in the placenta was analysed by RT-PCR in real time using primers obtained from RT² qPCR Primer Assay (200), (Qiagen; RefSeq Accession no.: NM_001275.3). The procedure was performed using RT² SYBR Green Mastermix according to the manufacturer’s guidelines: RT² SYBR Green Mastermix was combined with Qiagen CHGA primers and aliquated into the 96-well plate. Subsequently, proper amount of the cDNA of each sample, obtained in the previous step (2.2), was added into the wells RT-PCR was performed in a LightCycler 480 (Roche, Mannheim, Germany) device. The stability of the housekeeping gene candidates was analysed using Norm Finder software [13 (link)]. Each run was normalized using reference sample prepared from 160 pooled randomly-selected samples of first-strand cDNA from the study (80 samples) and control (80 samples) groups in equal volumes. The normalized gene expression was calculated according to Pfaffl [14 (link)].

cDNA was synthesized from 2000 ng of total RNA using a Maxima First Strand cDNA kit (Thermo Fisher Scientific, Warsaw, Poland). A RT-PCR reaction was performed with LightCycler 480 (Roche, Mannheim, Germany). Primers used for the reaction were commercially available and purchased from Qiagen (RT² qPCR Primer Assay (200); Qiagen; RefSeq Accession no.: NM_001275.3). The analysis was performed with 1.5 µL of 10× diluted cDNA with RT² SYBR Green Mastermix. The reactions were normalized with reference to the YWHAZ and GAPDH housekeeping genes, and a reference sample made from pooled samples (separate for each cell line). Normalized gene expression was calculated according to the Pfaffl equation [47 (link)].

After 4-h transfection and additional culture for 24 h in serum free DMEM-F12, 293 T and total ovaries were lysed using TRIzol reagent (catalog #15596026, Thermo Fisher Scientific) and RNA was extracted with Direct-zol RNA MiniPrep kit (#R2052, Zymo Research, Irvine, CA) following manufacturer’s protocol. High-Capacity cDNA Reverse Transcription Kit (#4368814, Thermo Fisher Scientific) was used to reverse transcribe 1 μg RNA. mRNA expression was quantified by q-RT-PCR amplification of cDNA using SYBR Green PCR Master Mix (#4309155, Thermo Fisher Scientific) and a Bio-Rad CFX384 Real-Time PCR Detection System. Q-RT-PCR was performed with primer assays from Qiagen RT² qPCR Primer Assay (200) (catalog #330001) targeting the Cyp17, Cyp19, Pgr, Ahm, Lhcgr, Fshr and, Foxl2 gene. Primer assay efficiencies were guaranteed by the manufacturer. Target gene expression was normalized on Gapdh and Actin expression.