Bio 65043 tetro cdna synthesis kit

Total RNA from the different cell lines was extracted with Trizol reagent and treated with DNase I to avoid potential contamination by genomic DNA. DNA-free RNA was reverse transcribed using Bioline (BIO-65043) tetro cDNA Synthesis Kit according to the manufacturer's instructions.
For semi quantitative RT-PCR 25 cycles were performed for the amplification of Actin, 35 cycles for for all the others. Primers for cDNA amplification were: GUCY1A2 for 5′GGTTACAGGGATGCAGAAAAGAAT3′; rev 5′TTCA GGGAGCTCTTTGCATAGG3′. NPR2 for 5′ACCATTAT CGTACCCTGGTT3′, rev 5′ TCTCGGTACGTGATCAC CAATA3′. PDE1A for 5′AGCAATGGTCTTTGCTGCTG; rev 5′AGTCGATAAGCTGCACTCAC. PDE3A for 5′CGT CACCTTCGCTAGTGAAA; rev 5′AACTCGTCTCAACA AGCCAG. ACTIN for 5′GCGAGAAGATGACCCA GATCA ; rev 5′CACAGGACTCCATGCCCAGGA. GUCY1B3 for 5′ GAGGAGTACAVACTAGGTTCCAGT; GUCY1B3 rev 5′ CACATGAAGCTCACATCATC; GUCY1A3 for AGACAGTAGACCTTCTGTGCTC; GUCY1A3 rev TCCACCTTGTAGACATCCAG.

Total RNA from germ cells was extracted with Trizol reagent and treated with DNase I to avoid potential contamination by genomic DNA. DNA-free RNA was reverse-transcribed using Bioline (Taunton, MA, USA; BIO-65043) tetro cDNA Synthesis Kit according to the manufacturer's instructions.
For semiquantitative RT-PCR, 25 cycles were performed for the amplification of Actin, 38 cycles for Cripto, Nodal and all others genes amplification from male germ cells. Densitometric analyses were performed by ImageJ (http://rsb.info.nih.gov/ij/index.html).
The Applied Biosystems (Foster City, CA, USA) 7300 Real-time PCR System and SsoAdvanced Universal SYBR Green Supermix (BIO-RAD 172-5271, Hercules, CA, USA) were used for quantitative RT-PCR (RT-qPCR). The comparative 2(-Delta Delta C(T)) method was used to determine the relative quantities of mRNA, using Gapdh mRNA as the endogenous normalizer. Sequences of oligonucleotides used for RT-PCR and RT-qPCR are given in Supplementary Table 1.