Abi prism 7900 system

To examine the CSF cytokines mRNA, the ABI PRISM 7900 System was applied (Applied Biosystems, Foster City, CA 94404, USA). As stated by the manufacturer's instructions, the mRNAs for IL-1β, IL-6, IL-8, and TNF-α expression were established, while 18S was designated as a standard control owing to its stable expression under the stimuli of SAH. Each sample was launched into a TaqMan Human Cytokine Card that enclosed probes and primers for specific targets. This procedure was performed via an affixed filling reservoir and a vacuum loading process via the ABI PRISM Card Filling Station.
Target mRNAs were standardized according to the reference gene (18S), and final data were expressed as a relative fold from the baseline. Comparative mRNA expression was set by the Livak and Schmittgen ΔCT method [40 (link)]. The results were analyzed if a 5-fold increase in the mRNA levels compared with the baseline to allow for data consistency.

Real-time RT-PCR was performed using the ABI Prism7900 System (Applied Biosystems - Life Technologies, Saint-Aubin, France) as described [10 (link)] with PCR primers for LZTS1 (Hs00232762_m1) (Applied Biosystems). The ∆∆CT method was used to quantify transcripts.

ChIP experiments were conducted according to a previous protocol^{49 (link)}. ChIP-re-ChIP was performed in MCF-7TamR cells as described previously^{50 (link)}. Briefly, protein-DNA complexes were eluted two times from primary immunoprecipitation (IP) in 20 mM DTT at 37 °C, 30 min/per elution, and diluted 1:50 in buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) followed by re-ChIP with second antibodies. The immunoprecipitated DNA was isolated and quantified by real-time PCR with SYBR Green using the ABI Prism 7900 system (Applied Biosystems). Primers used in this study are shown in Supplementary Table S5.

The qPCR was run using the ABI Prism7900 system (Applied Biosystems, Foster City, CA, USA) using the SYBR Green Real time PCR Master Mix (TOYOBO, Tokyo, Japan) according to the manufacturer’s protocol and the MIQE guidelines (http://www.rdml.org/miqe). Each 20 μL reaction consisted of 10 μL of SYBR Green Real time PCR Master Mix, 0.4 μL of Forward Primer (10 μM), 0.4μL of Reverse Primer (10 μM), 2 μL of cDNA and 7.2 μL of RNase-free H₂O. The reaction of qPCR was performed using the program: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 10 s and 72°C for 15 s. The qPCR products were verified with a melting curve at a range of 55–99°C. All samples were measured in triplicate to ensure reproducibility.

Total RNA was isolated from mGCs using the TRIzol Reagent (Invitrogen) according to the standard protocol. One μg RNA was reverse transcribed to cDNA with the PrimeScript RT Reagent Kit (Takara Biotechnology Dalian Co. Ltd., Dalian, China). qRT-PCR was performed on an ABI Prism 7900 system (Applied Biosystems, CA, USA) using the SYBR Premix Ex Taq (Takara Biotechnology Dalian Co. Ltd., Dalian, China). The following thermocycling protocol was used: denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 10 sec, annealing at 62°C for 20 sec, and extension at 72°C for 40 sec. The relative expression was calculated using the 2^−ΔΔCt method and normalized to GAPDH or U6. The sequences of primes are listed in Table 1 based on previous studies [22 (link)–24 (link)].

The CSF cytokines mRNA was examined via the ABI PRISM^® 7900 System (Applied Biosystems, Foster City, CA 94404, USA). According to the manufacturer’s instructions, the mRNAs for IL-1β, IL-6, IL-8, and MCP-1 expression were determined, while 18S was used as a standard control based on its stability. Primer sequences were employed for IL-1β: (242 bp: forward 5′-GCTCATCTGGGATCCTCTCC-3′ and reverse 5′-CCTGCCTGAAGCTCTTGTTG-3′); IL-6: (91 bp: forward 5′-GACAACTTTGGCATTGTGG-3′ and reverse 5′-ATGCAGGGATGATGTTCTG-3′); IL-8: (229 bp: forward 5′-TCTGCAGCTCTGTGTGAAGG-3′ and reverse 5′-ACTTCTCCACAACCCTCTGC-3′) and MCP-1: (457 bp: forward 5′-CTCTTCCTCCACCACTATGC-3′ and reverse 5′-CTCTGTCATACTGGTCACTTC-3′). Each sample was launched into a TaqM Each sample was launched into a TaqMan^® Human Cytokine Card that enclosed probes and primers for specific targets. This procedure was performed via an affixed filling reservoir and a vacuum-loading process via the ABI PRISM^® Card Filling Station. Final data were expressed as a relative fold from the baseline. Comparative mRNA expression was set by the Livak and Schmittgen ∆CT method [38 (link)]. The results were analyzed if a fivefold increase in the mRNA levels compared with the baseline to allow for data consistency.