Total cellular RNA was extracted using TRIzol reagent (Ambion, Carlsbad, CA). First-strand cDNA was synthesized from microRNA with a microRNA First-Strand cDNA Synthesis kit (Fulen Gene, Guangzhou, China). First-strand cDNA was synthesized from mRNA with a GoScript™ RT reagent kit (Promega, Beijing, China) according to the protocol provided by the manufacturer.
Goscript rt reagent kit
Manufactured by Promega
Sourced in China
The GoScript RT Reagent Kit is a reverse transcription (RT) kit designed for the synthesis of complementary DNA (cDNA) from RNA templates. The kit includes the necessary reagents and enzymes required for the RT reaction, allowing for the conversion of RNA to cDNA for subsequent use in downstream applications, such as qPCR or RT-PCR.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Lightcycler 480 pcr system, by Roche (2 mentions)
Trizol reagent, by Magen Biotechnology Co (1 mentions)
Sybr green qpcr mix, by GenStar (1 mentions)
Trizol regent, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex rt qpcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr mix, by Selleck Chemicals (1 mentions)
Gotaq qpcr master mix kit, by Promega (1 mentions)
5 protocols using goscript rt reagent kit
1
Comprehensive RNA Extraction and cDNA Synthesis
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from cells using the TRIzol reagent (Magen, R4801). As for tissue, the samples were grounded in liquid nitrogen before TRIzol extraction. Then, 1 μg of the total RNA was reverse transcribed into cDNA using a GoScript RT Reagent Kit (Promega, A5001), according to the manufacturer’s instructions. Real-time PCR was conducted with SYBR green qPCR Mix (GenStar, A301) on a LightCycler 480 PCR system (Roche). The quantitation of all target gene expression was normalized to the endogenous reference gene HPRT in PAMs or to GAPDH in Marc-145 cells via the 2−ΔCt method. The primer sequences that were used are listed in Table S1.
3
Total RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from cells using TRIzol regent (#335904; Invitrogen Corp., Carlsbad, CA, USA), and RNA was reverse transcribed to complementary DNA (cDNA) using a GoScript RT Reagent Kit (#A5001; Promega Corp., Fitchburg, WI, USA). We performed RT‐qPCR using a QuantStudio 6 Flex RT‐qPCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green PCR Mix (#B21703; Bimake, Houston, TX, USA). All RT‐qPCR primers are listed in Table S1 .
4
Gene Expression Analysis by qPCR
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According to indication of manufacture, extraction of Total cellular RNA by TRIZOL reagent (Ambion, Carlsbad, CA), Synthetism of First-strand cDNA was from mRNA with a Go Script™ RT reagent kit (Promega, Beijing, China). The mRNA expression levels of TGF-β1, Col1, α-SMA, and GAPDH (internal control) were analyzed by real-time PCR with a Go Taq ® qPCR Master Mix kit (Promega, Beijing, China). The data (after being normalized to GAPDH levels) were evaluated by the 2−ΔΔCt method. The primers are shown in Table 1 .
5
Quantifying PRRSV Infection in Marc-145 Cells
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Total RNA was isolated from Marc-145 cells using TRIzol reagent (Invitrogen) and then reverse transcribed into complementary DNA with a GoScript RT Reagent Kit (Promega) according to the manufacturer's instructions. To quantify the mRNA levels of EXT1 and PRRSV N, relative RT–qPCR was performed using a Light-Cycler 480 PCR system (Roche). The expression levels of target genes were normalized to the endogenous reference gene GAPDH using the 2−ΔΔCt method (where Ct is the threshold cycle). For the virus copies in infected Marc-145 cells, absolute RT–qPCR was conducted to quantify PRRSV M gene expression, and a standard curve was generated using a serially diluted plasmid containing the PRRSV M gene. The primers and TaqMan probe used for RT–qPCR are listed in Table S1 .
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