Clontech advantage rt for pcr kit

RNA was extracted as described previously [18 (link)] by using a PAXgene RNA kit (QIAGEN, Valencia, CA). After extraction, all RNA samples were tested for their integrity and concentration using a Bio-Rad Experion Automated Electrophoresis System (BIO-RAD, Hercules, CA). cDNAs were synthesized from total RNA extractions using a Clontech Advantage RT-for-PCR Kit (Clontech, Mountain View, CA). Prolactin expression analysis was conducted using a Bio-Rad IQ5 quantitative Real Time Polymerase Chain Reaction Detection System (BIO-RAD, Hercules, CA). GAPDH was used as an endogenous control. qRT-PCR conditions were as follows: 50°C for 2 minutes and 95°C for 10 minutes (95°C for 15 seconds, 62°C for 30 seconds, and 72°C for 30 seconds) for 47 cycles. Relative quantitation of prolactin and TNF-α mRNA expression was normalized to GADPH and fold changes were calculated using the 2^−ΔΔCT method. Primers utilized for prolactin, TNF-α, and GAPDH are listed below: 

Prolactin R: 5′ CGG CGC GGT CAA ACA GGT CT 3′.

 

Prolactin F: 5′ ACC AGG AAA AGG GAA ACG AAT GCC 3.

 

GAPDH-F: 5′ CCACCCATGGCAAATTCCATG 3′.

 

GAPDH-R: 5′ TCTAGACGGCAGGTCAGGTCC 3′TNF-alpha.

 

TNF-α-F: 5′-CTT CTC CTT CCT GAT CGT GG-3′.

 

TNF-α-R: 5′-GCT GGT TAT CTC TCA GCT CCA-3′.