Sybr green master mix

TRI reagent (Sigma-Aldrich, MO) was immediately added to TG and DRG tissue 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 GFAP Fwd: 5′-CCT TGA GTC CTT GCG CGG CA-3′, Rev: 5′-TTG GCC CTC CTC CTC CAG CC-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 an ABI 7000 Sequence Detection System (Applied Biosystems, CA). The GAPDH gene was used as an internal standard to which expression of other genes was normalized. Data were analyzed using the comparative C_t method, and compared with control values (Schmittgen and Livak 2008 (link)).

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)).