Cfx96 touch thermocycler

Based on previous studies, the rat TNC tissues, localized between − 14 and − 16 mm from bregma, and the VN tissues, localized between − 10 and − 12 mm from bregma, were extracted based on the rat brain atlas of Paxinos and Waston (6th edition) [16 (link), 30 (link)]. Total RNA from tissues was extracted using RNAiso Plus reagent (TaKaRa, Dalian) (21). RNA concentration and purity were quantified spectrophotometrically with NanoDrop (Thermo, USA). First-strand cDNA was generated using1 mg of total RNA with reverse transcriptase PrimeScript™ RT Reagent Kit (Takara, Dalian). The mRNA expression of rat CGRP was detected by qPCR withSYBR® Premix Ex TaqTM II (TaKaRa, Dalian) with a CFX96 Touch thermocycler (Bio-Rad, USA) according to the manufacturer’s recommendation. The reaction mixture (20 μL total) consisted of 10 μL 2× SYBR® mix, 8 μL nuclease-free water, 0.5 μL of primers (rat CGRP: F: 5′-GTTGGCTATTGTGCATCGTGTT-3′, R: CCGCTTGAGGTTTAGCAGAGTTA-3′; GAPDH, F: 5′-ATGACTCTACCCACGGCAAGCT-3′, R: 5′-GGATGCAGGGATGATGTTCT-3′) and 1 μL diluted cDNA. The reactions were performed as follows: an initial 5 min’s denaturation step, followed by 35 cycles of 95 °C for 30 s, 63 °C for 30 s, and 72 °C for 45 s, as well as a 7 min final extension step. Relative mRNA levels were calculated using the ΔΔCq method using GADPH mRNA as an internal control.

RT-qPCR was carried out as previously described [52 (link)] and summed up in Table 2. The TRI-Reagent (Sigma-Aldrich) was added to primary astrocyte samples to extract total mRNA. An equal amount of mRNA (1 μg) from each experimental group was quantified by a D30 BioPhotometer spectrophotometer (Eppendorf AG, Hamburg, Germany) and reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). RT-PCR reactions (20 μL) were set mixing specific primers, cDNA (20 ng) with the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and run in 96-well plates using the CFX96 Touch thermocycler (Bio-Rad, Hercules, CA, USA). All samples were run concurrently in triplicate. A two-step thermal protocol was used for 40 cycles (10 s at 95 °C and then 30 s at 60 °C) preceded by a polymerase activation step (3 min at 95 °C). Gene-specific amplification was controlled by including a melting curve analysis of the amplification products at the end of the reaction. The Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as reference gene to normalize each target amplicon. Data were analyzed using the Pfaffl method, correcting the delta Ct values for each primer efficiency [53 (link)]. All primer sequences are listed in Table 2.