Itaq universal sybrr green supermix

First-strand cDNA synthesis was performed with the iScriptTM cDNA Synthesis Kit (Bio-Rad #1708890) according to the manufacturer’s instructions. For qPCR reactions, the reaction mixture consisted of cDNA first-strand template, primers (500 nM each) and iTaqTM Universal SYBRR Green Supermix (Bio-Rad, #1725120). qPCR was performed in a BioRad CFX96 real-time system. Expression result was determined using the comparative Ct method (2^-ΔΔCt). Primers used are described in Additional file 9: Table S4. NbACT was used as the reference gene, using primers described in [23 (link)].

The expression level of selected differentially expressed mRNAs were validated by qRT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene. The primers for qRT-PCR are shown in Table S3. The qRT-PCR was performed on a Roche LightCycler^R 96 using iTaq^TM Universal SYBR^R Green Supermix (Bio-Rad, Hercules, CA, USA). The amplification protocol comprised one cycle at 95 °C for 5 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative gene expression was calculated by the 2^−∆∆Ct method with average cycle thresholds. Comparisons between the different groups from six independent samples from each group were statistically analyzed using the Student’s t-test (n = 6).

Total RNA was isolated using the TRIzol reagent (Life Technologies, U.S.A.). Five hundred nanograms of total RNA was reverse transcribed using the RETROScript cDNA synthesis kit (Life Technologies, U.S.A.).
Real-time PCR was performed using 7900HT Fast Real Time PCR by Applied Biosystems, the iTaqTM Universal SYBRR Green Supermix (Biorad) a Bio-Rad iQ iCycler detection system with SYBR green fluorophore. A melting curve analysis was made after each run to ensure a single amplified product for every reaction. All reactions were run at least in triplicate for each sample. Primers to study NGB expression were as follows NGB-Forward: 5′-TGGAAGACCTGTCCTCACTG-3′ and NGB-Reverse: 5′-GAGCAGAGACTCACCCACTG-3′ (Zhang et al., 2011 (link)). Gene expression was normalized using specific amplification of 18S.

mRNA expression of IRE-1α, XPB1s, PERK, ATF6, eIF2α, ATF4, TRAF2, ASK1, and CHOP were measured at the site of injury at 1 day (sham: n = 13, 6M/7F; TBI + saline: n = 12, 6M/6F; TBI + BPN: n = 13, 6M/7F), 3 days (sham: n = 10, 5M/5F; TBI + saline: n = 18, 8M/10F; TBI + BPN: n = 16, 8M/8F), and 7 days (sham: n = 11, 5M/6F; TBI + saline: n = 14, 8M/6F; TBI + BPN: n = 14, 5M/9F) post-injury. Brain tissues (approximately between bregma +2 mm and bregma −1 mm) were microdissected for RNA isolation as previously described.^{38 (link)} Total RNA was extracted using TRIzol (Sigma-Aldrich, St. Louis, MO) and were quantified using the Nanodrop ND-2000 Spectrophotometer (ThermoFisherScientific, Waltham, MA). Single-stranded complementary DNA (cDNA) was reverse transcribed from RNA using the High-Capacity cDNA Reverse Transcription Kit with RNase inhibitor (ThermoFisherScientific). Primers were custom designed (Table 1) and ordered from Integrated DNA Technology (Coralville, IA). Quantitative real-time polymerase chain reaction (qPCR) was performed with iTaq(tm) Universal SYBR(R) Green Supermix (Bio-Rad Laboratories, Hercules, CA), and the comparative threshold cycle method was used to assess differential gene expressions.^{38 (link)}