Superscript vilo kit

TRI reagent (MilliporeSigma, T9424) was used for RNA isolation from mouse liver and cell lines. RNA was reverse-transcribed using a SuperScript VILO kit (Invitrogen, Thermo Fisher Scientific). Gene expression was analyzed with the ABI Prism sequence detection system (SYBR Green; Applied Biosystems, Thermo Fisher Scientific). Gene-specific primers were synthesized by Thermo Fisher Scientific (Supplemental Table 4). Each sample was run in duplicate and normalized to Tbp (ChREBP-LKO cohorts), Ppib (HGFAC cohorts), or rplp0 for rat experiments.

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.

According to the manufacturer’s instruction, we extracted total RNA using Trizol Reagent (Ambion, Austin, TX, USA) and cDNAs with the Superscript Vilo kit (Invitrogen, Waltham, MA, USA). The reverse transcription reaction was performed at 42 °C for 30 min, at 99 °C for 5 min, and then cooled to 4 °C. We held the PCR mixture at 94 °C for 2 min and then cycled 30 times at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, followed by 10 min at 72 °C at the final cycle. We performed real-time PCR assays with TaqMan Universal PCR Master Mix and gene expression assays from Applied Biosystems (Waltham, MA, USA). We normalized gene expression with human gapdh as the endogenous control. We used the 2Δct method to analyze data and expressed data as arbitrary units or as fold difference.

The first strand cDNA synthesis was synthesized using SuperScript VILO kit (Invitrogen, Carlsbad, CA, USA). The qPCR was performed in Luna Universal qPCR master mix (New England Biolabs, Ipswich, MA, USA) using CFX96™ Optics Module (Bio-Rad, CA, USA). Relative gene expression was quantified and Gapdh was used as internal control. All primer sequences are listed in Supplementary Table S2.

Total RNA was extracted from spinal cord tissue 24 h following SCI using Trizol LS reagent (Invitrogen, Carlsbad, CA, United States), quantified with a spectrophotometer (NanoDrop Lite, Thermo Fisher) and 2.5 μg was reverse transcribed using the Superscript VILO kit (Invitrogen) in a volume of 20 μl. To evaluate gene expression 1 μl of cDNA was added to the EvaGreen qPCR Master Mix (Biotium, Inc., Fremont, CA, United States), in a volume of 20 μl per well and BAX (Fw: 5^′-CGAGCTGATCAGAACCATCA-3^′ Rv: 5^′-CTCAGCCCATCTTCTTCCAG-3^′), BCL2 (Fw: 5^′-ATACCTGGGCCACAAGTGAG-3^′ Rv: 5^′-TGATTTGACCATTTGCCTGA-3^′), Wnt3a (Fw: 5^′-TTCTTACTTGAGGGCGGAGA-3^′ Rv: 5^′-CTGTCGGGTCAAGAGAGGAG-3^′), DKK-1 (Fw: 5^′-AATCGAGGAAGGCATCATTG-3^′ Rv: 5^′-GCTTGGTGCATACCTGACCT-3^′) and β-catenin (Fw: 5^′-CGGCACCTTCCTATTTCTTCT-3^′ Rv:5^′-TCTGGAAATTAACTTCAGGCAAAC-3^′) were assayed. Samples were run in duplicate and GADPH (Fw: 5^′-GTCAAGGCTGAGAATGGGAA-3^′ Rv: 5^′-ATACTCAGCACCAGCATCAC-3^′) was used as housekeeping gene; the reaction was performed using the two-step thermal protocol as recommended by the manufacturer.
Results were calculated using the 2^-ΔΔC_t method, and expressed as n-fold increase in gene expression using the CTRL group as calibrator.

Total RNA was extracted from GF and EC cells for RTqPCR using Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA was reverse transcribed in a final volume of 20 μL using a Superscript VILO kit (Invitrogen). cDNA (1 μL) was added to the EvaGreen qPCR Master Mix (Biotium Inc., Fremont, CA, USA) (20 μL per well). The final primer concentration selected to perform the analysis was 10 μM. Samples were run in duplicate and β-actin was used as an endogenous control. Results were calculated using the 2^−ΔΔCT method and expressed as n-fold increase in gene expression using the CTRL group as the calibrator [21 (link)]. Primers used for targets and reference genes are listed in Table 1.