Quantitect primer assay

To measure doublecortin (DCX) mRNA levels in forebrain Ca_v1.2 cKO mice and AAV2-2/2-Cre-GFP injected cacna1c floxed (cacna1c^fl/fl) mice, mice were euthanized by rapid decapitation and whole brains were rapidly dissected. Brain tissue was sectioned on a 1 mm brain block. Dentate gyrus-containing tissue punches were obtained from forebrain Cav1.2 cKO and wild-type mice. For AAV2/2-Cre-GFP and AAV2/2-GFP injected mice, GFP goggles (BLS) were used to visualize GFP signal in brain sections containing the dentate gyrus and to selectively dissect GFP-positive tissue. Tissue punches were processed for total RNA isolation using the mirVana RNA isolation kit (Life Technologies) and cDNA was synthesized from purified RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). Cav1.2 mRNA levels were measured using cacna1c-specific primers (Qiagen QuantiTect Primer assay QT00150752), and DCX levels were measured using DCX-specific primers (Qiagen QuantiTect Primer assay QT02521155) on an ABI PRISM 7000 Sequence Detection System with SYBR Green PCR Master Mix (Applied Biosystems). Cycle threshold (Ct) values for target genes were normalized to the housekeeping gene gapdh (QuantiTect Primer assay QT01658692, Qiagen). Each experiment was performed in triplicate and values were averaged.

T cell effector genes were analysed on the same cDNA samples used for Treg signature gene analysis described above. qPCR was performed on a CFX96 real time system (BioRad) using QuantiTect Primer assays (Qiagen) for IL17-Rα, NFATc2, IL-21, RORγt, T-bet and IFNγ and SsoFast Evagreen Supermix (BioRad). Levels of Histone 3 and 18s were used to normalize target gene expression abundance (Histone: H3F3A BT020962, primers: fwd: 5′-ACTGGCTACAAAAGCCGCTC-3′, rev: 5′-ACTTGCCTCCTGCAAAGCAC-3′; 18 s: QuantiTect Primer assay, Qiagen).

HeLa or U2OS cells were stimulated with IFNα (180 U/mL). THP-1 cells were differentiated with PMA (200 ng/mL) for 24 h before stimulation with LPS (100 ng/ml). Total RNA was isolated using the High Pure RNA Isolation Kit (Roche) according to the manufacturer’s protocol. Reverse transcription was performed with 1 μg of RNA using the QuantiTect Reverse Transcription Kit (QIAGEN). ARTD8 and ARTD10 expression was analyzed by quantitative real-time PCR (qRT-PCR) using QuantiTect Primer Assays (QIAGEN). In each experiment, mRNA expression of the gene of interest was normalized to GUS (QuantiTect Primer Assays, QIAGEN).
For protein analysis cells were treated as above and harvested in RIPA buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 1% DOC; 0.1% SDS; PIC). Cellular debris was removed by centrifugation for 25 min at 4 °C. Lysates were fractionated using SDS-PAGE and analyzed by immunoblotting.

mRNA was isolated with NucleoSpin RNA isolation kit (Macherey-Nagel, #740955.250) according to the manufacturer’s instruction. Resulting RNA concentrations were determined with a NanoDrop ND-100 spectrometer. A total of 20 ng of RNA was used for each sample. One step SYBR Green-based reverse transcriptase PCR was accomplished using the 1 Step RT PCR Green ROX L Kit (highQu) following the manufacturer’s instruction. Specific fragments used for murine cell lines were (all Quantitect Primer Assays, Qiagen): ALDH1A3 (QT01077867), CXCR4 (QT00249305), Nestin (QT00316799), SOX2 (QT00249347), KCNN4 (K_Ca3.1, QT00105672). mRNA abundances were normalized to the geometric means of those of the housekeeper genes GAPDH (QT01658692) and PDHB1 (QT00163366). Fragments used for human cell lines were (again all Quantitect Primer Assays, Qiagen): KCNN4 (K_Ca3.1, QT00003780), ALDH1A3 (QT00077588), CXCR4 (QT00223188), NES (QT00235781) and SOX2 (QT00237601). mRNA abundances were normalized to the geometric means of those of the housekeeper genes GAPDH (QT01192646) and ACTB (QT00095431).
Measurements were conducted on a LightCycler480 (Roche), and crossing point values and melting curves were analyzed using LightCycler 480 software (Roche, version 1.5.0).

Total RNA was extracted from whole‐brain tissues using TRIzol (Invitrogen, Paisley, UK) according to the manufacturer's instructions. DNAse‐treated RNA samples were analyzed by quantitative RT‐PCR using QuantiFast™ SYBRVR green RT‐PCR kit (Qiagen Inc.) according to the manufacturer's instructions. All reactions were performed using a LightCycler (Roche, Mannheim, Germany). At the end of each PCR run, melting curve analysis was performed to verify the integrity and homogeneity of PCR products. Gene expression levels were calculated using standard curves for each gene, which were created by plotting threshold cycle (CT) values versus the logarithm of serial‐diluted RNA concentrations. A least‐square method was used for the determination of A and B values in the equation CT = A*Log (C_RNA) + B. The coefficient of determination (R²) was greater than 0.99. Values were normalized using the respective values for the housekeeping gene, Gapdh. All results were analyzed using the LightCycler software version 3.5 (Roche, Mannheim, Germany, RRID: rid_000088). QuantiTect Primer Assays were used for Il6 (Mm_Il6_1_SG), Il1b (Mm_Il1b_2_SG), tlr2 (Mm_Tlr2_1_SG), Csf1 (Mm_Csf1_2_SG), Lgals3 (Mm_Lgals3_1_SG), P2ry12 (Mm_P2ry12_3_SG), Nos1 (Mm_Nos1_2_SG), and Gapdh (Mm_Gapdh_3_SG), all QuantiTect Primer Assays from Qiagen.