Total RNA was extracted by using TRIzol (GIBCO Life Technology). First-strand cDNA was prepared from total RNA (1.5 μg) using the SuperScript® VILO cDNA Synthesis Kit and Master Mix (Thermo Fisher Technologies), and cDNA (1 μl) was amplified in triplicate using SYBR GreenER qPCR Supermix on an ABI Quantstudio® 3 Real-Time PCR instrument (Applied Biosystems). Primers for mouse Klf15, mouse Col1α1, mouse Vimentin, mouse Fibronectin, mouse c-Myc, and mouse Ctgf were designed using NCBI/Primer-BLAST (Supplemental Table 2 ). Light cycler analysis software was used to determine crossing points using the second derivative method. Data were normalized to housekeeping genes (ACTB) and presented as fold increase compared with RNA isolated from the control group using the 2−ΔΔCT method.
Abi quantstudio 3 real time pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in China
The ABI Quantstudio® 3 Real-Time PCR instrument is a thermal cycler designed for real-time polymerase chain reaction (PCR) analysis. It is capable of detecting and quantifying nucleic acids in real-time during the amplification process.
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Lab products found in correlation
2 protocols using abi quantstudio 3 real time pcr instrument
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Reverse Transcription and qRT-PCR Analysis of PEDV-Infected Piglet Liver
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The total RNA extracted from liver tissues of control and PEDV-infected piglets using the TRIzol Reagent (Invitrogen, MA, USA) was reverse-transcribed to single-stranded DNA (cDNA) using the HiScript II Q RT SuperMix (Vazyme biotech, Nanjing, China) according to the manufacturer's instructions. The purity and concentration of total RNA were evaluated by electrophoresis in 1% agarose gel and NanoReady Spectrophotometer (Suizhen, Hangzhou, China). The qRT-PCR analysis was carried out on an ABI QuantStudio 3 Real-Time PCR Instrument (Applied Biosystems) using the SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). The 10 μl reaction mixture contains 5 μl of AceQ qPCR SYBR Green Master Mix (2 ×),0.2 μl of ROX Reference Dye II (50 ×), 1 μl of cDNA template,0.2 μl of forward primer (10 μmol/L), and reverse primer (10 μmol/L), and 3.4 μl of ddH2O. The Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mRNA was detected as an internal reference to normalize the expression level of each transcript. The relative expression levels of indicated genes were calculated using the ΔΔCt method.
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