Nucleospin rna and protein purification kit

Total RNA was isolated using NucleoSpin RNA and Protein Purification Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA from each sample (2,000 ng) was reverse transcribed to cDNA using a Tetro cDNA Synthesis Kit (Bioline, London, UK) in a final volume of 20 µL. In order to evaluate the expression of type I LH-RH receptors and LH-RH ligand, primer sets were designed. Gene-specific primers for LH-RH-I receptor: sense 5′-GACCTTGTCTGGAAAGATCC-3′ (EXON 1 1,844–1,863), antisense 5′-CAGGCTGATCACCACCATCA-3′ (EXON 1 1,844–1,863), for LH-RH ligand: sense 5′-GGCCTTATTCTACTGACTTGG-3′, antisense 5′-TCTTCTGCCCAGTTTCCTCT-3′. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as an internal reference gene (sense 5′-GTATTCATTATAGTCAAGGGCATATCC-3′, antisense 5′-AGATGGTCAAGGTCGCAAG-3′). mRNA levels of LH-RH-R-I, LH-RH, and HPRT1 have been assessed by iQ™ SYBR^® Green Supermix (Bio-Rad Laboatories Inc, Hercules, CA, USA). Reactions were conducted according to the manufacturer’s protocol using MyiQ2 two-color real-time PCR detection system (Bio-Rad Laboratories Inc). All real-time amplifications were measured in triplicates. Results were evaluated with Bio-Rad iQ5 software (Bio-Rad Laboatories Inc) and changes in mRNA levels were calculated using the 2^40-Ct method.

Total protein was isolated using NucleoSpin RNA and Protein Purification Kit (Macherey-Nagel) according to the manufacturer’s instructions. Total protein amount of the supernatant was determined by a Nanodrop ND-1000 UV-Vis Spectrophotometer (ThermoFisher Scientific). Equal amounts of proteins (20 µg) were separated in 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene fluoride membrane using standard procedures.28 (link) Upon blocking with 5% BSA, membranes were incubated with primary antibodies (overnight, 4°C): anti-LH-RH-R, 1:200 dilution (sc-13944 rabbit polyclonal; Santa Cruz Biotechnology Inc), anti-GAPDH, 1:1,000 dilution (D16H11 rabbit monoclonal; Cell Signaling Technology, Danvers, MA, USA). Proteins were detected with anti-rabbit horseradish peroxidase conjugated antibody (mouse sc-2357; Santa Cruz Biotechnology Inc) and Luminata Forte Western horseradish peroxidase Substrate (Merck Millipore, Billerica, MA, USA). The protein bands were quantified using Image Lab software (Bio-Rad Laboratories Inc).