Super script 3 reverse transcriptase

Total RNA was extracted from the animal tissue using TRIzol⁴⁹ . First-strand cDNA was synthesized from a 1-µg sample using oligo (dt) primers (Sigma-Aldrich, MO, USA) and Superscript III reverse transcriptase. Real-time polymerase chain reaction (RT-PCR) was performed with Fast SYBR Green master mix in a 20-µL volume reaction using 7500 Fast RT-PCR system (Thermo Fisher Scientific, IL, USA). The following primers were used: for midkine, 5′-accgaggcttcttccttctc-3′ (forward), and 5′-ggctccaaattccttcttcc-3′ (reverse); for β-2 microglobulin (B2M), 5′-agcccaagaccgtctactgg-3′ (forward), and 5′-ttctttctgcgtgcataaattg-3′ (reverse). Gene expression levels were normalized to the level of B2M (Gene bank No: Midkine, NM_001012335.1; B2M, NM_009735).

Total RNA from cultured cells or tumor tissues was purified using Trizol (Invitrogen) and treated with DNase I (Sigma) according to the manufacturer's instructions. Oligo-dT or random hexamer were used to reverse transcribe 1 µg of RNA with SuperScript III Reverse Transcriptase (Sigma) according to the manufacturer's instructions. cDNA preparation and qRT-PCR analysis were performed as described previously [41] (link). For non-quantitative expression analysis, cDNA was amplified by PCR for 35 cycles at 60°C as annealing temperature and amplified products were visualized after electrophoresis in a 1–2% agarose gel.
Quantitative PCR was performed with a final 1 in 20 dilution of cDNA, 8 µM primers and Sybr Green MasterMix (Invitrogen). For normalization of transcript expression levels, human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcripts were used as internal controls. The average expression of internal control was used to calculate the relative gene expression.
Table S1 contains the list of primers used.

cDNA was synthesized using Superscript III Reverse Transcriptase (Sigma) according to the manufacturer’s instructions. qPCR was performed in 96-well plates using iQ™ SYBR Green Supermix (Bio-Rad) and an I-cycler iQ Real-Time PCR instrument (Bio-Rad). Measurements were performed in triplicate with a variability <0.5 Ct. mRNA expression was analysed by normalizing to that of the housekeeping gene GADPH. Primers were designed with PERL primer software using NCBI EntrezGene reference sequences as templates and synthesized by Sigma. The primers used are listed in Supplementary Table S4.

TriPure reagent (Sigma-Aldrich, MO, USA) was immediately added to brain tissue following dissection for mRNA extraction and stored at −80 °C. cDNA was synthesized using Super-Script III Reverse Transcriptase (Sigma-Aldrich, St. Louis, MO, USA). For real-time PCR analysis, duplicate cDNA samples were mixed with PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA, USA) and 125 nM forward and reverse primers of target genes (rat GAPDH Fwd: 5′-ACC CAG AAG ACT GTG GAT GG-3′, Rev: 5′-CAC ATT GGG GGT AGG AAC AC-3′; rat NOP Fwd: 5′-GTT CAA GGA CTG GGT GTT CAG CCA GGT AGT-3′; rat NOP Rev: 5′-TGC TGG CCG TGG TAC TGT CTC AGA ACT CTT-3′; rat preproN/OFQ Fwd: 5′-TGC ACC AGA ATG GTA ATG TG-3′, Rev: 5′-TAG CAA CAG GAT TGT GGT GA-3′ (Sigma-Aldrich) in a StepOnePlus Real-time PCR system (Applied Biosystems). The GAPDH gene was used as an internal standard to which the expression of other genes in each sample was normalized. Data were analyzed using the comparative Ct method. To determine fold change over sham, each 2^-DeltaCt value was divided by the average of all sham values [60 (link)], and groups were analyzed by one-way ANOVA (NOP receptor) or two-way ANOVA (preproN/OFQ) to determine the effects of genotype and/or injury severity.

TRI reagent (Sigma-Aldrich, MO) was immediately added to dissected tissues for mRNA extraction. cDNA was synthesized using Super-Script III Reverse Transcriptase (Sigma-Aldrich, MO). Real-time PCR was performed using SYBR Green Master Mix (AnaSpec, Fremont, CA) and 125 nM forward and reverse primers (rat TNF-α FWD: 5′-ACCACGCTCTTCTGTCTACTG-3′, REV: 5′-CTTGGTGGTTTGCTACGAC-3′, rat 28S: FWD: 5′-GAAGGCAAGATGGGTCACCA-3′, REV: 5′-GAACTTCCGTGGGTGACTCC-3′, rat GAPDH Fwd: 5′-ACCCAGAAGACTGTGGATGG-3′, Rev: 5′-CAC ATT GGG GGT AGG AAC AC-3′, rat NOP Fwd: 5′-GTT CAA GGA CTG GGT GTT CAG CCA GGT AGT-3′, rat NOP Rev: 5′-TGC TGG CCG TGG TAC TGT CTC AGA ACT CTT-3′, rat preproN/OFQ Fwd: 5′-TGC ACC AGA ATG GTA ATG TG-3′, Rev: 5′-TAG CAA CAG GAT TGT GGT GA-3′, all from Sigma-Aldrich) in QuantStudio StepOne qPCR (Applied Biosystems). The average of GAPDH and 28S CT values served as an internal standard to which expression of other genes were normalized. Data were analyzed using the comparative Ct method as described (34 (link)).