Separate cohorts of morphine, cocaine and vehicle abstinent animals were prepared for qRT-PCR experiments. Brains were removed and placed into a brain matrix (ASI Instruments, Warren, MI, USA). Bed Nucleus of the Stria Terminalis (BNST) and Central Nucleus of the Amygdala (CeA) were punched bilaterally out from 1mm-thick slices. Tissues were immediately frozen on dry ice and kept at -80°C until use. BNST and CeA punches from two mice were pooled in EA samples (n=5 samples/treatment). RNA was extracted and purified using the MIRNeasy mini-kit (Qiagen, Courtaboeuf, France). cDNA was synthetized using the first-strand Superscript II kit (Invitrogen®, Life Technologies, Saint Thomas, France) (Le Merrer et al., 2012 (link)). qRT-PCR was performed in quadruplets on a LightCycler 480 Real-Time PCR (Roche, Manheim, Germany) using iQ-SYBR Green supermix (Bio-Rad, Marnes-la-Coquette, France) kit with 0.25μl cDNA in a 12.5μl final volume. Gene-specific primers were designed using Primer3 software to obtain a 100- to 150-bp product (Table S1 ). Relative expression ratios were normalized to the level of actin and the 2–ΔΔCt method was applied to evaluate differential expression level. Primer sequences are displayed in Table S1 .
Superscript 2 first strand kit
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
The Superscript II First-strand kit is a reverse transcription kit used for the synthesis of first-strand cDNA from RNA templates. It contains the essential components required for the conversion of RNA to cDNA, including the SuperScript II reverse transcriptase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Lightcycler 480 real time pcr, by Roche (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Lightcycler 480 rt pcr, by Roche (1 mentions)
Syber green master mix, by Roche (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
7900 ht fast real time qpcr system, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna blood kit, by Macherey-Nagel (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
7 protocols using superscript 2 first strand kit
1
Quantifying Gene Expression in Addiction Regions
2
Dissection and qRT-PCR Analysis of Brain Regions
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Mice were killed by cervical dislocation. Brains structures (Nacc n = 8 D1R-CTL; n = 7 D1R-Gpr88 and CPu n = 9 D1R-CTL; n = 9 D1R-Gpr88, hippocampus: n = 9 D1R-CTL; n = 7 D1R-Gpr88 and amygdala: n = 6 D1R-CTL; n = 7 D1R-Gpr88) from D1R-Gpr88 and controls were quickly dissected out, frozen on dry ice and stored at −80°C until used. RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthetized using the first-strand Superscript II kit (Invitrogen, Life Technologies). Quantitative real-time PCR (qRT-PCR) was performed in triplicates on a LightCycler 480 RT- PCR (Roche) and SyberGreen masterMix (Roche). Thermal cycling parameters were 1 min at 95°C followed by 40 amplification cycles of 15 s at 95°C, 15 s at 60°C, and 30 s at 72°C. Relative expression ratios were normalized to the level of actin and the 2−ΔΔCt method was applied to assess differential expression level of GPR88.
3
Quantitative Real-Time PCR for Gene Expression
4
Robust RNA Isolation and cDNA Synthesis
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Total RNA was isolated from whole blood samples using the NucleoSpin RNA Blood kit (Macherey–Nagel, Düren, Germany). RNA integrity and its concentration were checked by 1% agarose gel and Nanodrop® ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, USA).
RNA reverse transcription was carried out as follows:
RNA reverse transcription was carried out as follows:
160 ng of total RNAs were reverse transcribed using both random hexamers and a specific GBA 3′ UTR primer (5′ GGCCTCCAGCCCCTG 3′) by using Superscript II First Strand kit (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions.
200 ng of total RNAs were reverse transcribed in 40 µl of final volume in a reaction mixture containing 4 µl TaqMan RT buffer 1×, 5.5 mM MgClq, 500 µM each dNTP, 2.5 µM random hexamers/specific GBA 3′ UTR primer, 0.4 U/µl RNase Inhibitor and 1.25 U/µl Multi-Scribe Reverse transcriptase provided by Applied Biosystems. The profile of the one-step reverse transcriptase was: 10 min at 25 °C, 30 min at 48 °C and 2 min at 95 °C.
5
Total RNA Extraction and RT-PCR Analysis
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Total RNA was harvested by TRIZOL reagent (Invitrogen) and reverse transcribed (SuperScript II First Strand kit; Invitrogen). Semiquantitative RT-PCR and qPCR were performed as described in our previous report [10 (link)].
6
Quantification of Inflammatory Markers in Bone Marrow
7
Quantitative Gene Expression Analysis
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RNA was extracted from exponentially growing cells using TRIzol reagent (Life Technologies). Total RNA was DNase-treated with RQ1 RNase-Free DNase (Promega). Reverse transcription was performed using Superscript II first strand kit (Invitrogen). qRT-PCR was performed with the SYBR Green PCR Master Mix on a StepOne Real-Time PCR System (Applied Biosystems). Primer sets used are listed in Supplementary Table 2 . mRNA levels were normalized using β-Actin mRNA levels.
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