Abi 7500ht real time pcr system

Total RNA from xenograft tumours and cells was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, tumours or cells were dissolved in 1 ml TRIzol^®, followed by total RNA extraction with 200 μl chloroform and RNA precipitation with 500 μl isopropanol. cDNA was synthesized from 1 μg total RNA using the PrimeScript RT reagent kit (Takara Bio, Inc.). Reverse transcription as performed as follows: 25°C for 5 min, 37°C for 30 min and 85°C for 5 sec. cDNA was diluted 20-fold with ddH₂O and used for qPCR with SYBR Premix EX Taq kit (Takara Bio, Inc.) in an ABI 7500HT real-time PCR system (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation, 95°C for 5 sec; followed by 35 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 25 sec and extension at 70°C for 30 sec. The gene levels for all samples were normalized to U6 small nuclear (sn)RNA (for miRNA) or GAPDH levels using the 2^−ΔΔCq method (22 (link)).

Total RNA was extracted from pituitary tissue using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. For this experiment, 4 μg of total RNA was treated with an M-MLV reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. cDNA was diluted with DRD2 Taqman^® gene expression assay (ID: Rn00561126_m1, DRD2, Thermo Fisher Scientific), RNAse-free water, and Taqman^® gene expression Master Mix (P/N 4369016, Thermo Fisher Scientific) using an ABI 7500 HT Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer’s recommendations. PCR was amplified in duplicates and three independent experiments using a Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Taqman^® gene expression assay (ID: Rn01775763_g1, GAPDH, Thermo Fisher Scientific) as endogenous controls. PCR amplification was performed in 96-well optical reaction plates for 40 cycles, with each cycle at 95°C for 15 seconds and 60°C for 1 minute. Fold changes were calculated using the 2^−ΔΔCt method.

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) was used to analyze tumor specimen. Total RNA was extracted using the TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers protocol. cDNA was synthesized from 2 µg total RNA using the M-MLV reverse transcriptase at 42°C for 1 h followed by 70°C for 10 min. GAPDH was used as the reference gene for RT-qPCR. The sequences used in the present study were ILK forward, 5′-GACGACATTTTCACTCAGTGCC-3′ and reverse, 5′-ACGGTTCATTACATTGATCCGTG-3′; GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse 5′-CACCCTGTTGCTGTAGCCAAA-3′. Gene-specific amplification of the 12 µl PCR mixture was performed using a ABI 7500HT real-time PCR system (Thermo Fisher Scientific, Inc.). The thermocycling condition were: 94°C for 3 min; followed by 22 cycles of 94°C for 30 sec, 94°C for 60 sec and 72°C for 60 sec; followed by a final extension step at 72°C for 5 min. DNA concentration was approximately doubled following each cycle of denaturation, annealing and elongation. The cycle quantification (Cq) of each sample was calculated, and the relative expression of the gene was calculated using the 2^−ΔΔCq method (13 (link)).