Verso cdna rt kit

Total RNA was extracted from snap-frozen samples using an RNeasy isolation kit (Qiagen, Valencia, CA, USA) with on-column DNase treatment (Qiagen). cDNA synthesis was performed on 1 μg of total RNA using a Verso cDNA RT kit (Thermo Scientific, Pittsburg, PA, USA); the protocols used were according to the manufacturer’s instructions. Real-time PCR was performed using an ABI 7500 Sequence Detection System (Applied Biosystems) in the presence of SYBR Green. Standard PCR conditions were used as prescribed in SYBR Green I core reagent protocol. PCR was performed using nucleotide primers of CD11b, CCR2, TNF-α, and IFNγ (obtained from Integrated DNA Technology, Coralville, IA, USA). Gene expression was calculated relative to the endogenous control sample (GAPDH) to determine relative expression values, using the 2^−ΔΔCt method (where Ct is the threshold cycle). All experiments were repeated three times.

Total RNA was extracted from the spinal cord (~5 mm surrounding the epicenter of the lesion site) of sham and SCI mice with a miRNeasy isolation kit (Cat# 74104, Qiagen). Complementary DNA (cDNA) was synthesized by a Verso cDNA RT kit (Cat# AB1453B, Thermo Scientific) per the manufacturer's protocol. Real-time PCR for target mRNAs was performed using TaqMan gene expression assays for Itgam (CD11b), Mm00434455_m1, TNFα, Mm00443258_m1; Csfr1, Mm01266652_m1; P2ry12, Mm00446026_m1; Trem2, Mm04209424_g1; Mfge8, Mm00500549_m1; Casp1, Mm00438023_m1; Casp6, Mm01321726_g1; Casp8, Mm01255716_m1; Bax, Mm00432051_m1; Bcl2, Mm00477631_m1; Gfap, Mm01253033_m1; Il-1a, Mm00439620_m1; Lrrc25, Mm00462019_m1; Gbp2, Mm00494576_g1; Osmr, Mm01307326_m1; Entpd2, Mm00515450_m1; Vim, Mm01333430_m1; C4a, Mm01132415_g1; GAPDH, Mm99999915_g1 (Applied Biosystems, Carlsbad, CA] on an QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). qPCR reactions were run in duplicates to eliminate mistakes of pipetting, which was composed of 3 stages, 50 °C for 2 min, 95 °C for 10 s for each cycle (denaturation), and finally, the transcription step at 60 °C for1 min. Gene expression was normalized by GAPDH and compared to the control sample to determine relative expression values by the 2-ΔΔCt method.

Total RNA was extracted from BMDMs and snap-frozen sham and TBI cortical tissue of WT and NOX2^−/− mice using an RNeasy isolation kit (Qiagen, Valencia, CA) with on-column DNase treatment (Qiagen). cDNA synthesis was performed using a Verso cDNA RT kit (Thermo Scientific, Pittsburg, PA); the protocols used were according to the manufacturer’s instructions. Real-time PCR was performed using TaqMan gene expression assays (Ym1, Mm00657889_m1; Arg1, Mm00475988_m1; SOCS3, Mm00545913_s1; IL-4Rα, Mm00446186_m1; TGF-β, Mm00441724_m1; SHIP1, Mm00494987_m1; and GAPDH Mm99999915_g1; Applied Biosystems, Carlsbad, CA) on an ABI 7900 HT FAST Real-Time PCR machine (Applied Biosystems). Samples were assayed in duplicate in one run (40 cycles), which was composed of 3 stages, 50 °C for 2 min, 95 °C for 10 s for each cycle (denaturation) and finally the transcription step at 60 °C for 1 min. Gene expression was calculated relative to the endogenous control sample (GAPDH) to determine relative expression values, using the 2 − ΔΔCt method (where Ct is the threshold cycle).

Total RNA was extracted from snap-frozen sham and TBI ipsilateral cortical and hippocampal tissue from young and aged mice (n=7-9/group) as described before. cDNA synthesis was performed using a Verso cDNA RT kit (Thermo Scientific, Pittsburg, PA) according to the manufacturer’s instructions. qRT-PCR was performed using TaqMan gene expression assays (Ifnb1, Mm00439552_s1; Irf7, Mm00516793_g1; Isg15, Mm01705338_s1; Ifi204, Mm00492602_m1; Irf1, Mm01288580_m1; Irf3, Mm00516784_m1; Irf4, Mm00516431_m1; Irf5, Mm00496477_m1; Irf7, Mm00516793_g1; Mx1, Mm00487796_m1; and Gapdh, Mm99999915_g1) on an ABI 7900 HT FAST Real Time PCR machine (Applied Biosystems, Carlsbad, CA). Samples were assayed in duplicate in one run (40 cycles), composed of 3 stages: 50°C for 2 minutes, 95°C for 10 seconds for each cycle (denaturation), and finally the transcription step at 60°C for 1 minute. Gene expression was calculated relative to the endogenous control sample (Gapdh) to determine relative expression values, using the 2−ΔΔCt method (where Ct is the threshold cycle) (43 (link)).

Total RNA was extracted from the perilesional ipsilateral cortex and hippocampus of sham and CCI mice with a miRNeasy isolation kit (Cat# 74104, Qiagen, Valencia, CA). Complementary DNA (cDNA) was synthesized by a Verso cDNA RT kit (Cat# AB1453B, Thermo Scientific, Pittsburg, PA) per the manufacturer's protocol. Real‐time PCR for target mRNAs was performed using TaqMan gene expression assays for cybb (NOX2), Mm01287743_m1; Cyba (p22phox) Mm00514478_m1; ITGAM (CD11b), Mm00434455_m1; TNFα, Mm00443258_m1; Il1b (IL‐1β), Mm00434228_m1; IL‐6, Mm00446190_m1; chil3 (Ym1), Mm00657889_mH; Arginase‐1 (Arg1), Mm00475988_m1; IL‐4Rα, Mm01275139_m1; TGFβ, Mm01178820_m1; IL‐10, Mm0‐0439614_m1; SOCS3, Mm01342740_g1; Hvcn1 (Hv1), Mm01199507_m1; GAPDH, Mm99999915_g1 (Applied Biosystems, Carlsbad, CA] on an QuantStudio™ 5 Real‐Time PCR System (Applied Biosystems). Samples were assayed in duplicate in 1 run (40 cycles), which was composed of three stages, 50°C for 2 min, 95°C for 10 s for each cycle (denaturation), and finally, the transcription step at 60°C for1 min. Gene expression was normalized by GAPDH and compared to the control sample to determine relative expression values by the 2−ΔΔCt method.

RNA samples were obtained after sample processing with the RNeasy mini kit (Cat# 74104, Qiagen) from 5 mm of spinal cord tissue at various time-points post-injury. Complementary DNA (cDNA) was synthesized by a Verso cDNA RT kit (Cat# AB1453B, Thermo Scientific) per the manufacturer’s protocol. Real-time PCR for target mRNAs was performed with TaqMan gene expression assays for Itgam (Mm00434455_m1), CD83 (Mm01350412_m1), P2yr12 (Mm01950543_s1), Tmem119 (Mm00525305_m1), Trem2 (Mm04209424_g1), Gfap (Mm01253033_m1), Gdf15 (Mm00442228_m1), Opalin (Mm00463365_m1), Fcrls (Mm01219428_m1), Cxcl10 (Mm00445235_m1), Fkbp5 (Mm00487406_m1), H2-T23 (Mm00439246_g1), Csf1r (Mm01266652_m1), Pllp (Mm00452740_m1), Ennp6 (Mm00624107_m1), Plekhm1 (Mm00805590_m1).