Total RNAs were extracted from the prefrontal cortex, and then isolated with Trizol reagent. BioRT cDNA First Stand synthesis kit(Bioer technology, Hangzhou, China) was used to synthesize cDNA. RT-PCR was performed using LightCycler® 480 SYBR Green PCR Master Mix (Roche,USA) and analyzed using LightCycler® 480 SYBR Green SoftwareII. The expression of respective genes was normalized to that of glyceraldehyde phosphate dehydrogenase (GAPDH) within the same sample. The primers used were as follows: 5′-CGTGTCTGCACCTAGCCTCTATC-3′ and 5′- GCGAAACCAGGTCAGGATTC-3′ (IκBα), 5′-CAGGACCAGGAACAGTTCGAA-3′ and 5′-CCAGGTTCTGGAAGCTATGGAT-3′ (NF-κB-p65), 5′-TCGGTTGCATGAAGGC-3′ and 5‘- GGTTTTCTTCGTTGGGC-3′ (BDNF), 5′-GAATGATGCTGGGCAAGAGA-3′ and 5′-CAGTTGGCTTCTGGTGTTC-3' (Iba-1), 5′-TACAGGGCCTGCAGACATTAACCA-3′ 5′-ATTCTCTTGCTGCCTCCCTGTTCT-3′ (CREB), 5′-ATGTGTCCGTCGTGGATCTGA-3′ and 5′- ATGCCTGCTTCACCACCTTCT-3′ (GAPDH). RT-PCR was first identified to be specific using the melting curves and the relative expression of each mRNA level was calculated with the 2−ΔΔCT method after normalizing Ct (cycle threshold) values with GAPDH.
Lightcycler 480 sybr green pcr master mix
Manufactured by Roche
Sourced in United States
The LightCycler 480 SYBR Green PCR Master Mix is a ready-to-use reagent designed for real-time PCR applications. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence during amplification. The master mix also includes all necessary components for efficient PCR amplification, including DNA polymerase, buffer, and dNTPs.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Vazyme (2 mentions)
Lightcycler 480 sybr green softwareii, by Roche (1 mentions)
Biort cdna first stand synthesis kit, by Bioer (1 mentions)
Trizol protocol, by Thermo Fisher Scientific (1 mentions)
4 protocols using lightcycler 480 sybr green pcr master mix
1
Quantitative Analysis of Gene Expression
2
Quantitative Analysis of Gene Expression
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Total RNAs were extracted from cultured cells using the TRIzol reagent (Thermo Fisher Scientific Inc., Shanghai, China), and cDNA was synthesized using the M-MLV reverse-transcriptase (Vazyme Biotech Co., Ltd., Nanjing, China). In the reaction of reverse transcription, 1 μg of total RNA was mixed with 0.5 μg/μl of oligo(dT) primer, followed by incubation at 65°C for 5 min and 4°C for 2 min. The tubes were then immediately incubated at 42°C for 30 min, and then chilled at 4°C. For qPCR, cDNA was amplified using gene specific primers and the Lightcycler 480 SYBR Green PCR Master Mix (Roche, Basel, Switzerland) on the Lightcycler 480 instrument (Roche). The conditions used for qPCR were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed in triplicate. The results were analyzed using the 2–ΔΔCt method and normalized using β-actin. Primer sequences for qPCR were as follows: β-actin (5′-TTCCTTCCTGGGCATGGAGT and 5′-TCTTCATTGTGCTGGGTGCC), YY1 (5′-CCCACGGTCCCAGAGTCCA and 5′-GTGTGCGCAAATTGAAGTCC), and FOXM1 (5′-GCAGGCTGCACTATCAACAA and 5′-TCGAAGGCTCCTCAACCTTA).
3
Quantitative PCR Analysis of Cellular RNA
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Total cellular RNA was extracted using the TRIzol protocol (Thermo Fisher Scientific Inc., Shanghai, China) and subsequently analyzed by reverse transcription followed by quantitative PCR (RT-PCR). In the reaction of reverse transcription, we added 1 μg of RNA and 0.5 μg/μL of oligo(dT) primer, followed by incubation at 65 °C for 5 min and 4 °C for 2 min. The tubes were then immediately incubated at 42 °C for 30 min and 4 °C. The quantitative PCR analyses were performed using the LightCycler 480 SYBR Green PCR Master Mix and the primers listed in Table S1 in the Roche Lightcycler 480 instrument. The data for each gene were normalized against GAPDH levels.
4
Quantitative Real-Time PCR Analysis
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Total RNAs were extracted from cultured cells or tissues using the TRIzol reagent (Thermo Fisher Scientific, 15596026). Two μg of the isolated RNAs were reverse-transcribed into cDNA with oligo(dT) primers and M-MLV reverse-transcriptase (Vazyme, R021-01) according to the manufacturer’s instructions. The Light-Cycler 480 SYBR Green PCR Master Mix (Roche, S4438) and the primers listed in Supplementary Table 3 were used in qPCR to analyze the cDNA in each group on the Lightcycler 480 instrument (Roche, Basel, Switzerland). All data were normalized against β-actin levels and calculated using the 2–ΔΔCt method.
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