One microgram of total RNA, isolated from macrophages using the PrepEase RNA/Protein Spin kit (Affymetrix), was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real-time PCR was performed using an ABI Prism 7900 real-time PCR instrument (Applied Biosystems) with the QuantiFast SYBR Green PCR kit (Applied Biosystems). The primer sequences used are as follows: Mrc1, 5′-GGTTGCTATCACT-CTCTATGC-3′ (forward) and 5′-TTTCTTGTCTGTTGCCGTAGTT-3′ (reverse); CCL5, 5′-CCAGCAGTCGTCTTTGTCAC-3′ (forward) and 5′-CTCTGGGTTGGCACACACTT-3′ (reverse); and SDF1, 5′-ATTCTCAACACTCCAAACTGTGC-3′ (forward) and 5′-ACTTTA-GCTTCGGGTCAATGC-3′ (reverse). Data are displayed as expression ratios of target genes normalized to hypoxanthineguanine phosphori-bosyltransferase (HPRT). Quantitative real-time PCR data were analyzed by the 2−ΔΔ;Ct method (37 (link)).
Abi prism 7900 real time pcr instrument
Manufactured by Thermo Fisher Scientific
The ABI Prism 7900 is a real-time PCR instrument manufactured by Thermo Fisher Scientific. It is designed to perform quantitative polymerase chain reaction (qPCR) experiments. The instrument uses fluorescence detection to monitor the amplification of DNA or RNA targets in real-time during the PCR process.
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Lab products found in correlation
Quantifast sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
Krt10, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
2 protocols using abi prism 7900 real time pcr instrument
1
Quantitative PCR analysis of macrophage genes
2
Quantitative PCR Analysis of Skin Biopsy
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The skin biopsy specimens of each mouse in TRI Reagent® (Sigma-Aldrich Inc) were homogenized, and chloroform was added. After centrifugation, the aqueous phase was transferred to a new tube and isopropanol was added. After centrifugation, the supernatant was removed and 70% ethanol was added to the RNA pellet. RNeasy Mini Kits (Qiagen, Valencia, CA, USA) were used according to the manufacturer’s protocol to isolate RNA from keratinocytes or the skin biopsies of mice. The RNA was reverse transcribed into cDNA using SuperScript® VILOTM MasterMix according to the manufacturer’s protocol (Life Technologies). The cDNA was analyzed using real-time quantitative-PCR and an ABI prism 7900 real-time PCR instrument (Applied Biosystems, ThermoFisher Scientific) as described previously.20 (link) Primers and probes for GAPDH, IL-4, IL-5, IL-10, IL-12, IL-13, IL-33, transforming growth factor (TGF)-β, FLG, LOR, transglutaminase (TGM)-3, keratin (KRT)-1, KRT-10, and desmoglein (DSG)-1 were purchased from Applied Biosystems and normalized to the GAPDH mRNA levels.
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