Total RNA was isolated from the stomach mucosa of animals using TRIzol (Invitrogen, CA, USA), according to the manufacturer’s recommendations. The extracted total RNA was purified with DNase treatment using a DNA-free kit as a template (Ambion, USA) to generate first-strand cDNAs using a high-capacity cDNA Reverse Transcription kit (Applied Biosystems, USA). Quantitative real-time PCR was performed using a StepOne Plus Instrument (Applied Biosystems) with specific Taqman qRT-PCR primers and probes (Table 2 ). For analysis, the FNDC5 gene expression levels were normalized using the mRNA expression of two known housekeeping genes as recommended by the guidelines of MIQUE (The Minimum Information for Publication of Quantitative Real-Time PCR Experiments)22 (link), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and eukaryotic 18S rRNA (18S) (TaqMan: Applied Biosystems). The delta-delta-Cp-method with the geometric mean of the two reference genes for normalization was performed23 (link). Results are expressed as fold-changes in experimental group compared to a control group.
Taqman qrt pcr primers
Manufactured by Thermo Fisher Scientific
TaqMan qRT-PCR primers are oligonucleotide sequences designed for use in reverse transcription and quantitative real-time PCR assays. They are intended to amplify and detect specific target sequences in RNA samples.
Automatically generated - may contain errors
2 protocols using taqman qrt pcr primers
1
Quantitative Real-Time PCR for FNDC5 Gene
2
NUCB2 Gene Expression Analysis
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Total RNA was isolated from the stomach mucosa of animals using TRIzol (Invitrogen, CA, United States), according to the manufacturer’s recommendations. The extracted total RNA was purified with DNase treatment using a DNA-free kit as a template (Ambion, United States) to generate first-strand cDNAs using a High-capacity cDNA Reverse Transcription kit (Applied Biosystems, United States). Quantitative real-time PCR was performed using a StepOne Plus Instrument (Applied Biosystems) with specific Taqman qRT-PCR primers and probes. For analyses, the NUCB2 gene expression levels (Assay ID: Rn01510621_m1) were normalized to the mRNA expression levels of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) (Assay ID: Rn01527840_m1, TaqMan: Applied Biosystems) and are expressed relative to the average value of the control group.
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