For real-time RT-PCR analysis of gene expression, cMPs and BMDMs were collected and total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. cDNA was synthetized using qScript cDNA Supermix (Quanta, Gaithersburg, MD) and real-time PCR analysis was performed on the 7900HT Fast Real-Time PCR instrument (Applied Biosystems, Grand Island, NY) using FAM-labeled gene-specific probes and primers (Applied Biosystems). The level of target gene expression was calculated as 2−ΔCt where ΔCt = Cttarget − CtGAPDH with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous control and Ct indicating threshold cycle.
Fam labeled gene specific probes and primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
FAM-labeled gene-specific probes and primers are fluorescent oligonucleotides designed for use in various molecular biology techniques, such as real-time PCR and in situ hybridization. They are used to detect and quantify specific genetic sequences within a sample.
Automatically generated - may contain errors
3 protocols using fam labeled gene specific probes and primers
1
Real-Time PCR Gene Expression Analysis
2
RNA Extraction and qPCR Analysis
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Total RNA was extracted from lung tissue or isolated cells using RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed into cDNA using qScript cDNA Supermix (Quanta Biosciences, Beverly, MA). Real-time PCR reactions were performed using FAM-labeled gene-specific probes and primers (all from Applied Biosystems, MA, USA) on a 7900HT Sequence Detection System with TaqMan primer sets for GAS6, TYRO3, AXL, MERTK, CXCL1, CXCL2, TNF, IL-6 and GAPDH transcript levels as described in the online Supplementary Methods
3
Real-Time PCR Gene Expression Analysis
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