Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to evaluate the expression of liver SOD, CAT and GPX genes in tilapias. Used primer sequences were illustrated in Table 1 . The total RNA was extracted from tissue using the RNeasy Kit (Qiagen, Hilden, Germany) following the company's protocol. The Reverse Transcript kit (Qiagen, Hilden, Germany) was used cDNA synthesis. The qPCR of the β actin (a housekeeping gene) and studied genes were performed using a Qiagen QuantiTect SYBR Green PCR kit in a Rotor-Gene Q cycler (Qiagen, Hilden, Germany).
Reverse transcript kit
Manufactured by Qiagen
Sourced in Germany
The Reverse Transcript Kit is a laboratory equipment product designed for the reverse transcription of RNA into complementary DNA (cDNA). The kit contains the necessary reagents and enzymes to facilitate this process, enabling the conversion of RNA samples into a more stable and versatile form for further analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Rotor gene q cycler, by Qiagen (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green qrt pcr kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green qpcr kit, by Qiagen (1 mentions)
3 protocols using reverse transcript kit
1
Evaluating Antioxidant Gene Expression in Tilapia Liver
2
Isolation and Quantification of miRNA
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Total RNA was isolated from cultured cells and tissues using the TRIzol reagent (Life Technologies, Grand Island, NY, USA). Total RNA was isolated from the culture medium (CM) of cultured cells and from serum using the TRIzol LS (Life Technologies) reagent according to the manufacturer's protocol. Reverse transcription of miRNA was performed by using the Reverse Transcript Kit (QIAGEN, Duesseldorf, Germany). For gene expression analysis, qRT-PCR was performed using the miScript SYBR Green qRT-PCR kit (QIAGEN). β-actin (ACTB) and U6 were used as an endogenous control for cultured cells and tissue samples. Fold changes were calculated using the 2ΔΔCt method. The compilation of data was transformed using base 2 logarithmic transformation. After the transformation, the data closely followed normal distribution. For CM and serum samples, a standard curve was made for the endogenous control and quantification. All reactions were performed in triplicate. All of the oligonucleotides and primers used in the experiments are listed in Tables S1 and S2.
3
Quantifying miRNA Expression in Dendritic Cells
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Total RNA of DCs was extracted using TRIzol (Life Technologies, New York, USA) reagent according to the manufacturer's protocol. Reverse transcription was performed using Reverse Transcript Kit (QIAGEN, Duesseldorf, Germany). qPCR was performed with miScript SYBR Green qPCR kit (QIAGEN, Dusseldorf, Germany). miR-1228 amplification was performed as was negative control [38] . Beta-actin were amplified as a reference (Fig. 5A). Fold changes were calculated using the 2 -ΔΔCt method. All reactions were performed in triplicate. The used primers are listed in Table 2.
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