Total RNA was extracted using RNAiso plus reagent (Takara) according to the manufacturer's instructions. First-strand complementary DNA was synthesized from 1 μg of RNA using the RT mix kit (Takara). Real-time PCR was performed using SYBR Green real-time PCR analysis (Biotech). Expression of β-Actin was used to standardize the samples, and the results were expressed as a ratio relative to control. Primer sequences used for quantitative PCR were shown in Table S4 .
Rt mix kit
Manufactured by Takara Bio
Sourced in China
The RT mix kit is a laboratory product designed for reverse transcription (RT) reactions. It contains the necessary components to convert RNA into complementary DNA (cDNA) for downstream applications. The kit includes a reverse transcriptase enzyme, reaction buffer, and other essential reagents required for the RT process. The core function of the RT mix kit is to enable the conversion of RNA to cDNA, which is a crucial step in various molecular biology and genomics workflows.
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Lab products found in correlation
2 protocols using rt mix kit
1
Quantitative Gene Expression Analysis
2
LAMP and Turbidity for FAdV Detection
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The DNAs or RNAs of FAdVs (1–7) and the control strains (9–16) were extracted according to the instructions of the DNA/RNA extraction kit (BioTek Co., China). RNA specimens were reverse-transcribed to cDNA using the RT Mix Kit (Takara, China).
The LAMP kit was obtained from Eiken Chemical Co., Japan. The LAMP reaction system (25 μL) contained 12.5 μL of 2 × buffer, 1 μL of enzyme mixture, FIP and BIP (40 pmol each), F3 and B3 (10 pmol each), LB and LF (20 pmol each) and 2 μL of the template. The mixture was placed at 63 °Cfor 60 min, and ddH2O was used as the control. Three replicates of each sample were run for LAMP and real-time PCR.
Real-time turbidimetry and Tris-EDTA visual reagent (TVR, patent-pending, JXD Co., China) were used to evaluate the reaction results. White precipitates of Mg2P2O7 were produced during the LAMP reaction, and thus, the turbidity of the reaction tube was monitored every 6 s by a real-time turbidimeter (La-320, Eiken Chemical Co., Japan). A curve was then drawn to judge whether the reaction was positive. The addition of the TVR indicator to the reaction system resulted in different colors. Once double-chain nucleic acids appear in the amplification system, the dye groups can bind to them and show a bright green color, indicating a positive result; a colorless solution is deemed a negative result.
The LAMP kit was obtained from Eiken Chemical Co., Japan. The LAMP reaction system (25 μL) contained 12.5 μL of 2 × buffer, 1 μL of enzyme mixture, FIP and BIP (40 pmol each), F3 and B3 (10 pmol each), LB and LF (20 pmol each) and 2 μL of the template. The mixture was placed at 63 °Cfor 60 min, and ddH2O was used as the control. Three replicates of each sample were run for LAMP and real-time PCR.
Real-time turbidimetry and Tris-EDTA visual reagent (TVR, patent-pending, JXD Co., China) were used to evaluate the reaction results. White precipitates of Mg2P2O7 were produced during the LAMP reaction, and thus, the turbidity of the reaction tube was monitored every 6 s by a real-time turbidimeter (La-320, Eiken Chemical Co., Japan). A curve was then drawn to judge whether the reaction was positive. The addition of the TVR indicator to the reaction system resulted in different colors. Once double-chain nucleic acids appear in the amplification system, the dye groups can bind to them and show a bright green color, indicating a positive result; a colorless solution is deemed a negative result.
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