Quantinova sybr green real time pcr kit

Cells were collected, and total RNA extracted using the RNeasy Plus Mini Kit (Qiagen, Milan, Italy). The RNA concentration was determined using Nanodrop spectrophotometer ND-1000 (Thermofisher), and 2 μg of RNA were reverse transcribed using Superscript-VILO kit (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR was performed from 100 ng/sample of cDNA with Rotor-Gene Q using Qiagen QuantiNova SYBR Green Real Time-PCR Kit. Primers are listed in Table 1. Melting curve analysis confirmed the specificity of the amplified products. Data were analyzed applying the ΔΔCt method and expressed as a fold change vs. control.

After cell collection, total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Milan, Italy). RNA concentration was detected with Nanodrop spectrophotometer ND-1000 (ThermoFisher) and 2 µg of RNA were reverse transcribed using Superscript-VILO kit (ThermoFisher) according to the manufacturer’s instructions. qRT-PCR was performed on a 1:100 dilution of the reverse transcription reaction per sample, using the Rotor-Gene Q and Qiagen QuantiNova SYBR Green Real Time-PCR Kit. Primers used were Hs_BDNF_1_SG QuantiTect Primer Assay (QT00235368) and Hs_RPLP0_1_SG QuantiTect Primer Assay (QT00075012), both from Qiagen. RPLP0 was used as the endogenous control for normalization. Melting curve analysis confirmed the specificity of the amplified products. Data were analyzed applying the ∆∆Ct method and expressed as fold change versus control.

Real-time RT-PCR was carried out with the Rotor-Gene Q (Qiagen, Germantown, MD, United States). The amplification reaction mix included the Master Mix Qiagen (10 µL) (Qiagen QuantiNova SYBR Green Real-Time PCR Kit, Germantown, MD, United States) and cDNA (1 µL,100 ng). Forty-five amplification cycles were carried out for each sample. Results were analyzed with the 2^−ΔΔCt method (Leggio et al., 2019 (link)). Quantitative PCR experiments followed the MIQE guidelines. Gene expression levels were normalized with levels of housekeeping gene (18S). Primers were purchased from Eurofins Genomics (Milan, Italy) and Qiagen (Milan, Italy). Forward and reverse primer sequences (for human and mouse genes) and catalogue numbers are herein listed: human IL-1β (Forward: 5′-AGC​TAC​GAA​TCT​CCG​ACC​AC-3'; Reverse: 5′-CGT​TAT​CCC​ATG​TGT​CGA​AGA​A-3′), human IL-6 (Catalogue Number QT00083720), human TNF-α (Forward 5′-AGC​CCA​TGT​TGT​AGC​AAA​CC-3'; Reverse 5′-TGA​GGT​ACA​GGC​CCT​CTG​AT-3′), human 18S (Forward 5′-AGT​CCC​TGC​CCT​TTG-3'; Reverse 5′-GAT​CCG​AGG​GCC​TCA​CTA​AAC-3′), human BDNF (Catalogue Number QT00235368), mice 18S (Forward: 5′-GTT​CCG​ACC​ATA​AAC​GAT​GCC-3′; Reverse: 5′-TGG​TGG​TGC​CCT​TCC​GTC​AAT-3′), mice BDNF (Forward: 5′-GTT​CGA​GAG​GTC​TGA​CGA​CG-3′; Reverse: 5′-AGT​CCG​CGT​CCT​TAT​GGT​TT-3′), and mice IL-6 (Cat. No. QT00098875).