Superscript 2 reverse transcription first strand cdna synthesis kit

After isolation of RNA with BioRad Total RNA Extraction from Fibrous and Fatty Tissue kit (BioRad), cDNAs were generated from 200 ng RNA with the Superscript II Reverse Transcription First Strand cDNA Synthesis kit (Invitrogen). 10 ng RNA was combined with primers [5′-ATTGGACATAAGGGCGACAA-3′ and 5′-AGTCTCCTTTGGCTCCTGGT-3′ for Col19a1 (Su et al., 2016 (link)) or 5′-GGACCAGAGCGAAAGCATTTG-3′ and 5′- GCCAGTCGGCATCGTTTATG-3′ for 18S] and the iQ SYBRGreen Supermix (BioRad). Quantitative real-time PCR (qPCR) was performed on a Chromo 4 Four Color Real-Time system (BioRad) with 1 cycle of 95°C for 30 s and 40 cycles of amplification (95°C for 5 s, 60°C for 30 s, 55°C for 60 s, read plate) and a melting curve analysis. Relative quantities of RNA were determined using the ΔΔ-CT method (Livak and Schmittgen, 2001 (link)). At least three samples (each in triplicate) were examined for each age.

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)).