7500 fast rt pcr machine

RNA from frozen hypothalamic tissue was extracted using TRIzol Reagent (Thermo Fisher) and subsequently reverse transcribed into cDNA with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the manufacturer’s instructions. AgRP and NPY gene expression was measured using TaqMan Gene Expression Master Mix (Thermo Fisher) and a 7500-Fast RT-PCR machine (Applied Biosystems). As Siberian hamster TaqMan primers and probes are not commercially available, custom primer/probe sets were generated (Applied Biosystems) based on previously described Siberian hamster AgRP and NPY sequences [45 ]. AgRP primers/probe- forward primer: AGGCCCTGCTGCAGAAG, reverse primer: GACTCGCGATTCTGTGGATCTAG, reporter probe: ACCTCCGCCAACGCT; NPY primers/probe- forward primer: CTCCGCTGGTGCATCCT, reverse primer: GTGCTGGCTGAGGGATACC, reporter probe: AAGCCTGACAATCCTG. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) was used as an endogenous control as its expression was consistent across all animals and treatments (A.T. and T.J.B. unpublished observations).

Different variants of RET kinase domain were designed and ordered from Geneart (Life Technologies). RET variants were expressed in SF21 cells and harvested 72h post transfection. Subsequently proteins were purified and phosphorylated. For determining the protein thermal shift protein variants were incubated with DMSO or 1µM compound. Sypro-Orange dye (Life Technologies) was added to each drug treatment and thermal shift was measured in a 7500 Fast RT PCR machine (Applied Biosystems) in a temperature range of 25 – 90°C. Subsequent analysis was performed using Protein Thermal Shift Software v1.2 (Applied Biosystems).

RNA extraction was conducted with TRIzol (Thermo Fisher Scientific, 15596026). cDNA synthesis was done by using the First Strand cDNA Synthesis Kit (Applied Biosystems, 4368814) according to the manufacturer’s instructions. Real-time PCR was performed with 2× SYBR Green (Applied Biosystems, 4368706) on an ABI 7500 fast RT PCR machine. Primers used were as follows: mouse—Gapdh: 5′-CGT CCC GTA GAC AAA ATG GT-3′, 5′-TTG ATG GCA ACA ATC TCC AC-3′; Il1b: 5′-GAT CCA CAC TCT CCA GCT GCA-3′, 5′-CAA CCA ACA AGT GAT ATT CTC CAT G-3′; Il6: 5’-GAC AAA GCC AGA GTC CTT CAG AGA G-3′, 5′-CTA GGT TTG CCG AGT AGA TCT C-3′; Tnf: 5’-CAT CTT CTC AAA ATT CGA GTG ACA A-3′, 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′; Nlrp3: 5’-TCA GAT TGC TGT GTG TGG GAC TGA-3′, 5′- AGC TCA GAA CCA ATG CGA GAT CCT-3′; Ifna: 5’-GCT AGG CHY TRT GCT TTC CT-3′, 5′-CAC AGR GGC TGT GTT TCT TC-3′; Ifnb: 5’-GCC TTT GCC ATC CAA GAG ATG C-3′, 5′-ACA CTG TCT GCT GGT GGA GTT C-3′.
Human—GAPDH: 5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′, 5′-AGC CTT CTC CAT GGT GGT GAA GAC-3′; IL6: 5’-GCC TTC GGT CCA GTT GCC TT-3′, 5′-GCA GAA TGA GAT GAG TTG TC-3′; TNF: 5’-ATG ACT TCC AAG CTG GCC GT-3′, 5′-TCC TTG GCA AAA CTG CAC CT-3′; IL1B: 5’-CCA CAG ACC TTC CAG GAG AAT G-3′, 5′-GTG CAG TTC AGT GAT CGT ACA GG-3′; IFNG: 5’- GAG TGT GGA GAC CAT CAA GGA AG-3′, 5′- TGC TTT GCG TTG GAC ATT CAA GTC-3′.