Itag sybr green supermix

RNA from cerebral cortex (Ctx; P21), dorsal lateral geniculate nucleus (dLGN; of the dorsal thalamus; P21), hippocampus (P0, P7, P15, P21, P60), superior colliculus (P21), olfactory bulb (P21) and retina (P21) (n = 3, each pooled from 3 to 5 mice) was isolated using the Fibrous and Fatty Tissue RNA extraction kit (BioRad). cDNAs were generated from 250 ng RNA with Superscript II Reverse Transcription First Strand cDNA Synthesis kit (Invitrogen). Quantitative real-time PCR (qPCR) was performed on a CFX Connect Real-Time system (BioRad) using iTag SYBRGreen Supermix (BioRad) by the following primers: col25a1, 5′- GAT TCT CCT CTT CGG CCT CT -3′and 5′- AAA TAA GAA CGG CCA GGG AG -3′; col23a1, 5′- GCA ATC AGG ACG AGA TGG CT-3′ and 5′- AAA GTC TCC CGG TGT ACC CT-3′; col17a1, 5′-TGG GAT CAG CTT TGG GCA TC-3′ and 5′- GAC AAA CCA GCG GCT CGG A-3′; col13a1, 5′- AAG GGA GAA GCA GGC CTA GAG-3′ and 5′-TGG AGT ACC AGG CAA TCC CAG-3′; gapdh, 5′-CGT CCC GTA GAC AAA ATG GT-3′ and 5′-TTG ATG GCA ACA ATC TCC AC-3′. The following cycling conditions were used with 12.5 ng of cDNA: 95°C for 30 s, followed by 40 cycles of amplification (95°C for 5 s, 60°C for 30 s, 55°C for 60 s) and a melting curve analysis. Relative quantities of RNA were determined using the 2^−ΔCT method (Livak and Schmittgen, 2001 (link)).

mRNA expression analyses were performed using our previously published methods (Luk et al., 2021 (link)). Briefly, muscle samples were analyzed for intramuscular gene expression of DRP1, OPA1, MFN1, PINK1, and Parkin at BL, 12 and 24 h post‐exercise using real‐time PCR, which was performed using # 2 ABI PRISM70500 Sequence Detection System (Applied Biosystems) with iTag SYBR Green Supermix (Bio‐rad) and was normalized to Human beta 2‐microglobulin (B2 M). Relative fold change in transcript abundance was determined using the 2^−ΔΔCt method.

Two-week-old Arabidopsis seedlings were collected and immediately frozen in liquid nitrogen. Total RNA was isolated using the Total RNA I solation Kit (Qiagen) and treated with RNase-free DNase I (Qiagen). For real-time PCR assays, 1 μg of total RNA was used for first strand cDNA synthesis using oligo (dT) primers and Iscript reverse transcriptase (Bio-Rad) according to the manufacturer’s protocol. Quantitative expression assays were carried out with iTag SYBR Green Supermix (Bio-Rad) and an ABI PRISM7000 Sequence Detection System (Applied Biosystems). Arabidopsis ACTIN2 was used as a reference to normalize the expression of target genes. Each experiment was repeated three times with three technical repeats for each.