Primepcr probe assay

After performing reverse transcription, quantitative PCR analysis was performed both LightCycler 480 II (Roche, Basel, Switzerland) with FastStart Essential DNA Probes Master (Roche) as previously described (13 (link), 14 (link)) and CFX Opus Real-Time PCR Systems (Bio-Rad, Hercules, California, USA) with PrimePCR™ Probe Assay. Primer sequences for LightCycler 480 II were as follows: Arg1 sense, 5′-gaatctgcatgggcaacc-3′; Arg1 antisense, 5′-gaatcctggtacatctgggaac-3′; Chi3l3 sense, 5′-aagaacactgagctaaaaactctcct-3′; Chi3l3 antisense, 5′-gagaccatggcactgaacg-3′; Fizz1 sense, 5′-ccctccactgtaacgaagactc-3′; Fizz1 antisense, 5′-cacacccagtagcagtcatcc-3′; Il12p40 sense, 5′-ttgctggtgtctccactcat-3′; Il12p40 antisense, 5′-gggagtccagtccacctcta-3′; Tnfα sense, 5′-ctgtagcccacgtcgtagc-3′; Tnfα antisense, 5′-ttgagatccatgccgttg-3′; Actb sense, 5′-aaggccaaccgtgaaaagat-3′; Actb antisense, 5′-gtggtacgaccagaggcatac-3′. Unique assay ID of PrimePCR Probe (BioRad) for CFX Opus Real-Time PCR Systems were as follows: STAT3: qDreCIP0044562, Socs1: qMmuCEP0057945.

RNeasy microkit (Qiagen) was used according to the manufacturer's instructions to extract total RNA from dissected LGs of animals ranging from E15 to adult (34w). cDNAs were generated from biological triplicates by using the SuperScript™ III Reverse Transcriptase kit (for RT PCRs, Invitrogen) or the QuantiTect Reverse Transcription Kit (for multiplex PCRs, Qiagen, 205310), according to the provider's recommendations.
Subsequently to cDNA synthesis, reverse transcription PCRs for Aquaporin 1, 5, and 8 were performed using an annealing temperature of 60°C for 40 cycles. One hundred fifty nanograms of total RNA was used for each reaction.
The primer sequences are given in the following table:
Multiplex qRT-PCRs (CFX96 Touch™ Real-Time PCR Detection System, Bio-Rad) were performed using iTaq universal probe super mix (Bio-Rad, 1725130). Ten nanograms of cDNA were used per reaction. Probe combinations (PrimePCR Probe Assay, Bio-Rad):

Combination 1: GAPDH-Cy5 (qMmuCEP0039581); Krt14-Hex (qMmuCEP0058885); Acta2-Cy5.5 (qMmuCIP0032840); Krt19-FAM (qMmuCIP0033699).

Combination 2: GAPDH-Cy5 (qMmuCEP0039581); Notch2-Hex (qMmuCIP0030263); Hey1-Tex615 (qMmuCEP0057542).

Combination 3: GAPDH-Cy5 (qMmuCEP0039581); Hey1-Tex615 (qMmuCEP0057542); Krt14-Hex (qMmuCEP0058885); Acta2-Cy5.5 (qMmuCIP0032840).

Gene expression levels were normalized using GAPDH expression levels.

Prefrontal cortex tissues were extracted 24 h after the PPI test and immediately stored at −80 °C (n = 8–9 (short-term) and n = 6–10 (long-term)). The RNA was purified with the RiboPure™ KIT (Invitrogen) and the reverse transcription was performed with 1μg of total RNA and the SuperScript™ II Reverse Transcriptase (Invitrogen). PCR reactions were conducted using PrimePCR™ Probe Assay (Bio-Rad) to quantify mRNA levels of glutamic acid decarboxylase, 67 kDa isoform (GAD67) (ID: qMmuCEP0060617), brain derived neurotrophic factor (BDNF) (ID: qMmuCEP0058759), synaptophysin (SYP) (ID: qMmuCIP0035577), CB1R (ID: qMmuCEP0038879) and CB2R (ID: qMmuCEP0039299). To evaluate postsynaptic density protein 95 (PSD95) (ID: 4453320), TaqMan™ Gene Expression Assay (Applied Biosystems™) was used. GAPDH (ID: qMmuCEP0039581) expression was used as endogenous control gene for normalization. PCR assays were carried out with the CFX Connect Real-Time PCR Detection System (Bio-Rad). The fold changes in gene expression of JWH-018 treated animals in comparison with controls were calculated using the 2^−ΔΔCt method.

A total of three genes related to bone metabolism and mineralization were included in the present study: Runx2 (ID: 860), SP7 (ID: 121340), and ALPL (ID: 249). One-step real-time quantitative polymerase chain reaction (RT-qPCR) was performed by using the Reliance Multiplex RT-qPCR Supermix combined with the following Prime PCR probe assays (BioRad, Hercules, CA, USA): Runx2 (qHsaCEP0051329), SP7 (qHsaCEP0025867), and ALPL (qHsaCEP0053252). As suggested by Abuna et al. [56 (link)], Eif2b1 (qHsaCIP0030434) and YWHAZ (qHsaCIP0029093) were selected as reference genes, considering a wide range of algorithms for assessing expression stability in osteoblasts. Assays were conducted in a CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA), and the thermal cycling conditions were 50 °C (10 min) for reverse transcription, 95 °C (10 min) for DNA polymerase activation and template denaturation, and 40 cycles at 95 °C (10 s) and 60 °C (30 s) in a 10 µL reaction volume, according to manufacturer’s recommendations. For each biological sample, a total of three technical replicates were included. Multiplexing was validated experimentally after comparing the Cq values and amplification efficiencies of multiplex and singleplex reactions. For RT-qPCR efficiency calculation, five 5-fold serial dilution assays were performed, and the standard curve method was applied [57 (link)].