Total RNA was extracted using TRIzol reagent (Life Technologies China, Inc.). The amount and purity of the RNA were determined on a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). One microgram of isolated RNA was used to synthesize first-strand cDNAs using the ReverTra Ace-a First-strand cDNA Synthesis Kit (TOYOBO, Japan). Real-time PCR was performed on a Roche LightCycler 480 using the SYBR Green I Kit (TOYOBO, Japan) according to the manufacturer’s instructions. Elongation factor-1α, β-actin, and 18s were used as internal controls. Relative mRNA levels were determined using the standard ΔΔcycle threshold method.
Lightcycler 480
Manufactured by Toyobo
Sourced in Japan
The LightCycler 480 is a real-time PCR instrument designed for high-throughput nucleic acid analysis. It features a 96-well plate format and supports various fluorescence-based detection methods, including SYBR Green, hydrolysis probes, and melting curve analysis. The LightCycler 480 provides precise temperature control and uniform heating for accurate quantification and genotyping of DNA and RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Sybr green mix, by Toyobo (3 mentions)
Dntps, by Roche (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Reverse transcription system, by Promega (2 mentions)
Sybr green realtime pcr master mix, by Toyobo (2 mentions)
Sybr green pcr kit, by Toyobo (2 mentions)
Revertra ace α first strand cdna synthesis kit, by Toyobo (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 kit, by Toyobo (1 mentions)
Pd n 6, by Cytiva (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Nucleospin rna column kit, by Takara Bio (1 mentions)
Random primer, by Promega (1 mentions)
Mirna rt pcr kit, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr green dye, by Toyobo (1 mentions)
Revertra ace, by Toyobo (1 mentions)
12 protocols using lightcycler 480
1
RNA Extraction and qRT-PCR Analysis
2
Transcriptional profiling of Streptomyces gilvosporeus
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Total RNA isolation and RT-qPCR were performed as described previously (47 (link)). Mycelia of S. gilvosporeus F607 and S. gilvosporeus DO7 were harvested from fermentation liquid at 24, 72, 120, 168, and 216 h and then rapidly frozen in liquid nitrogen. Total RNA was extracted from mycelia using an RNA extraction kit (SBSBIO, Beijing China) according to the manufacturer’s protocol and then treated with Turbo DNA-free reagents (ABI Ambion, Austin, TX, USA) to remove residual chromosomal DNA. cDNAs were synthesized using random hexamer primers (pdN6, Amersham Pharmacia Biotech, Buckinghamshire, England), M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and dNTPs (Roche, Basel, Switzerland). RT-qPCR assays were performed on a Roche Light Cycler 480 instrument using SYBR Green Mix (Toyobo, Osaka, Japan). Relative quantities of cDNA were normalized to the amounts of the 16S rRNA gene. For RT-qPCR assays, experiments were conducted in triplicate. The primers used are listed in Table S3.
3
Quantifying Fungal Gene Expression
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Total RNA isolation and real-time quantitative PCR (RT-qPCR) procedures were performed as described previously (Fu et al., 2017 (link)). Mycelium of S. clavuligerus F613-1 and ΔcagRS were harvested from fermentation liquid at 24, 72, 120, 168, and 216 h, rapidly frozen in liquid nitrogen and then total RNA was extracted using an RNA extraction kit (SBSBIO, Beijing China) according to the manufacturer’s protocol. Total RNA samples were treated with Turbo DNA-free reagents (Ambion, United States) to remove the residual chromosomal DNA. The cDNAs were synthesized using random hexamer primers (pdN6, Amersham Pharmacia Biotech, England), M-MLV reverse transcriptase (Invitrogen, England) and dNTPs (Roche, Switzerland). Real-time PCR assays were performed on the Roche LightCycler 480 using SYBR Green Mix (ToYoBO, Osaka, Japan). Relative quantities of cDNA were normalized to the amounts of 16S rRNA. For RT-qPCR assays, experiments were conducted in triplicate. Statistical analysis was performed using SPSS Statistics V19.0 software.
4
Quantitative RT-PCR for FDX1 Gene
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Total RNA was extracted using TRIzol reagent (TIANGEN BIOTECH Co., Ltd., Beijing, China), and 1 µg of RNA was subjected to reverse transcription with the ReverTra Ace™ qPCR RT Kit (TOYOBO, Osaka, Japan). Real-time quantitative PCR was performed in a LightCycler 480 with QuantiNova SYBR Green dye (TOYOBO, Osaka, Japan). The primers used in this study are listed as follows:
FDX1 forward, 5′-TTCAACCTGTCACCTCATCTTTG-3′;
FDX1 reverse, 5′-TGCCAGATCGAGCATGTCATT-3′.
FDX1 forward, 5′-TTCAACCTGTCACCTCATCTTTG-3′;
FDX1 reverse, 5′-TGCCAGATCGAGCATGTCATT-3′.
5
RNA Extraction and qPCR Analysis
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After a 48‐hour incubation period, cells from the direct and indirect coculture were harvested, and total RNA was extracted using a NucleoSpin RNA column kit (Takara Bio, Kusatsu, Japan). Reverse transcription and real‐time polymerase chain reaction analyses were done using a Light‐Cycler 480 with a Thunderbird SYBR quantitative polymerase chain reaction mix kit (Toyobo, Osaka, Japan) as previously described.19 The forward and reverse primer sets are presented in Table S1 . Data were analyzed using the standard curve method. The cycle threshold was calculated using the default settings of the real‐time sequence detection software (ThermoFisher Scientific). Three independent experiments were conducted in each CF line.
6
Quantitative Real-Time PCR Assay
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Total RNA was extracted from breast tissue specimens or cells using the Trizol reagent (Invitrogen) following manufacturer's instructions. The cDNA was generated with random primers (Promega) using the Reverse Transcription System (Promega) or miRNA RT-PCR Kit (Takara, Shiga, Japan). Real-time PCR was carried out on a Roche LightCycler480 using SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan). PCR reactions were run in triplicate for three independent experiments. U6 snRNA or β-actin was used as an internal control. The sequences of PCR primers are listed in Supplementary Table S1.
7
RT-qPCR Quantification of Gene Expression
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Total RNA from aerial parts of four-week-old plate-grown plants was used for reverse-transcription (RT) with MMLV reverse transcriptase according to the manufacture’s protocol (Promega). Genes selected and corresponding primers were shown in Additional file 3 . Real-time quantitative PCR was performed on Roche Light Cycler 480 using the SYBR green PCR kit (TOYOBO, Osaka, Japan) and PCR was conducted according to the following protocol: 15 s denaturation at 94°C, 15 s annealing at 57°C, 15 s elongation at 72°C in 40 cycles. Fluorescence was detected at 80°C. Samples were analyzed in triplicate using independent cDNA samples and were quantified by the comparative cycle threshold method [55 (link)].
8
Quantitative RNA Expression Analysis
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Total RNA from MM cells was isolated using the Trizol reagent (Takara, Shiga, Japan) following manufacturer’s instructions. The obtained RNA was reverse-transcribed to synthesize the complementary DNA using random primers and the Reverse Transcription System (Promega, Madison, WI, U.S.A.). Real-time PCR was carried out on a Roche Light Cycler480 using SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). The relative quantitation of each gene was calculated using 2−ΔΔCT.
9
Transcriptional profiling of Streptomyces gilvosporeus
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Total RNA isolation and RT-qPCR were performed as described previously (47 (link)). Mycelia of S. gilvosporeus F607 and S. gilvosporeus DO7 were harvested from fermentation liquid at 24, 72, 120, 168, and 216 h and then rapidly frozen in liquid nitrogen. Total RNA was extracted from mycelia using an RNA extraction kit (SBSBIO, Beijing China) according to the manufacturer’s protocol and then treated with Turbo DNA-free reagents (ABI Ambion, Austin, TX, USA) to remove residual chromosomal DNA. cDNAs were synthesized using random hexamer primers (pdN6, Amersham Pharmacia Biotech, Buckinghamshire, England), M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and dNTPs (Roche, Basel, Switzerland). RT-qPCR assays were performed on a Roche Light Cycler 480 instrument using SYBR Green Mix (Toyobo, Osaka, Japan). Relative quantities of cDNA were normalized to the amounts of the 16S rRNA gene. For RT-qPCR assays, experiments were conducted in triplicate. The primers used are listed in Table S3.
10
Quantitative PCR Analysis of GhGT26 Expression
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Quantitative PCR was conducted with the primers GhGT26P1 and GhGT26P2 to analyze the expression patterns of GhGT26 in cotton seedlings under various treatments from the wild and transgenic plants. The G. hirsutum ubiquitin7 (U7F, U7R) and Arabidopsis ACT2 (ACT2F, ACT2R) genes were used separately as the standard controls. Primers used in this experiment are listed in Supplementary Table S1 . Real-time PCR was performed on a Roche Light Cycler 480 using the SYBR Green PCR Kit (TOYOBO, Osaka, Japan). The PCR mix was composed of 10 μL SYBR qPCR Mix, 2 μL cDNA, 0.5 μL of each primer (10 mM), and 7 μL PCR grade water in a final volume of 20 μL. The PCR reactions were carried out according to the following conditions: 1 cycle of 95 °C for 30 s; 40 cycles at 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 15 s; and fluorescence was detected at 80 °C. Each sample was analyzed in triplicate, and the expression levels were calculated using the 2−ΔΔCT comparative CT method [47 (link)].
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