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.
Biorad total rna extraction from fibrous and fatty tissue kit
Manufactured by Bio-Rad
The Bio-Rad Total RNA Extraction from Fibrous and Fatty Tissue kit is a laboratory product designed to efficiently extract total RNA from fibrous and fatty tissue samples. The kit provides a standardized protocol and reagents to facilitate the RNA extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Chromo 4 four color real time system, by Bio-Rad (2 mentions)
Superscript 2 reverse transcription first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Superscript 2 reverse transcriptase first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using biorad total rna extraction from fibrous and fatty tissue kit
1
Quantitative Real-Time PCR for Gene Expression
2
Quantitative Real-Time PCR of RNA Transcripts
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RNA was isolated using the 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). Quantitative real-time PCR (qPCR) was performed on a Chromo 4 Four Color Real-Time system (BioRad) using iQ SYBRGreen Supermix (BioRad; Su et al., 2010 (link)). Col19a1 primers for qPCR were 5′-ATTGGACATAAGGGCGACAA-3′ and 5′-AGTCTCCTTTGGCTCCTGGT-3′. Gapdh primers for qPCR were 5′-CGTCCCGTAGACAAAATGGT-3′ and 5′-TTGATGGCAACAATCTCCAC-3′. Syt2 primers for qPCR were 5′-CTGCCTGGTTTACAGAGCAA-3′ and 5′-TGTTTCTCATGGTGGCAGAG-3′. qPCR primers were designed over introns. The following cycling conditions were used with 10 ng RNA: 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, read plate) and a melting curve analysis. Relative quantities of RNA were determined using the ΔΔ−CT method. A minimum of n = 3 experiments (each in triplicate) was run for each gene, at each age examined. To be considered differentially expressed, genes had to be twofold higher in the averaged sample sets (n = 3, P < 0.05). Each individual run included separate GAPDH control reactions.
3
Transcriptional Profiling of Math5 in Retina and dLGN
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Retina and dLGN tissue were harvested from C57/BL6 mice at different embryonic and postnatal ages using methods described elsewhere [51 (link)]. RNA was isolated using the Bio-Rad Total RNA Extraction from Fibrous and Fatty Tissue kit (Bio-Rad). Reverse transcription and cDNA generation were made using Superscript II Reverse Transcriptase First- Strand cDNA Synthesis kit (Invitrogen). The following math5 primer pairs were used: 5′- ATGGCGCTCAGCTACATCAT- 3′ and 5′-GGGTCTACCTGGAGCCTAGC- 3′.
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