Total RNA was extracted with TRIzol reagent (Thermo Fisher, United States), and 1 μg RNA was reverse-transcribed into cDNA using oligo dT primer and M-MLV reverse transcriptase (Promega, United States). Quantitative RT-PCR was used using a Fast SYBR Green Master Kit and Light Cycler 480 system (Roche, Switzerland) according to the manufacturer’s instructions. The expression level of each target gene was normalized to GAPDH and measured by the comparative CT method (ΔΔCT). Primer sequences used are listed in Table 1 .
Fast sybr green master kit
Manufactured by Roche
Sourced in Switzerland
The Fast SYBR Green Master Kit is a qPCR reagent designed for the rapid and sensitive detection of target DNA sequences. It contains a proprietary SYBR Green I dye for fluorescent labeling and a hot-start DNA polymerase for increased specificity. The kit is suitable for a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Lightcycler 480 system, by Roche (5 mentions)
M mlv reverse transcriptase, by Promega (4 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Random primer, by Promega (1 mentions)
Pcr master mix, by Promega (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Labworks v4, by Analytik Jena (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
6 protocols using fast sybr green master kit
1
RNA Extraction and qRT-PCR Analysis
2
Quantitative Real-Time PCR for Gene Expression
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For cDNA synthesis, 500 ng of RNA was reverse transcribed using M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Real-time PCR was performed using the Fast SYBR Green Master Kit and LightCycler 480 system (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The expression level of each gene was normalized to the expression of GAPDH. Primer sequences were summarized in Table 1 .
3
Quantitative gene expression analysis
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Total RNA was extracted with Trizol reagent (Thermo Fisher, USA), and 1 μg of RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase and oligo(dT) or a random primer (Promega, USA). Quantitative real-time polymerase chain reaction (RT-PCR) was performed using a Fast SYBR Green Master Kit and Light Cycler 480 system (Roche, Switzerland) according to the manufacturer's instructions. The expression level of each target gene was normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and measured using the comparative CT method (ΔΔCT). PCR master mix (Promega, USA) was used for conventional RT-PCR to detect the splicing isoforms of XBP1, NCOR2 and DNAJC10. The primer sequences used are listed in Supplementary Table S1 .
4
Quantitative Gene Expression Analysis
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Total RNA was extracted from cells using TRIZOL reagent (Invitrogen, Grand Island, NY), and 1000 ng of RNA was reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI). Real-time PCR was performed using the Fast SYBR Green Master Kit and Light Cycler 96 system (Roche, Basel, Switzerland), and the expression level of each gene was normalized to the expression of the housekeeping gene GAPDH. The primer sequences are listed in Additional file 2 : Table S2.
5
Total RNA Extraction and Real-Time PCR
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Total RNA was extracted using Trizol reagent (Invitrogen, Grand Island, NY). The total RNA was reversely transcribed using M-MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. The real-time PCR was performed using the Fast SYBR Green Master Kit and Light Cycler 480 system (Roche, Basel, Switzerland) according to the manufacturer's instructions. Primers are listed in the Supplementary Table S2 .
6
Quantitative Gene Expression Analysis
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The total RNA was isolated from cells with TRIzol Reagent (Sigma Aldrich) according to the standard procedure. For cDNA synthesis, 2000 ng of RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen) following the manufacturer's instructions. qRT-PCR was performed using the Fast SYBR Green Master Kit and LightCycler 480 system (Roche, Basel, Switzerland) according to the manufacturer's instructions. The expression level of each target gene was normalized to the expression of GAPDH and measured by the comparative Ct (2 -ΔΔCt ) method. The results were expressed as log10 (2 -ΔΔCt ). Each sample was analyzed in triplicate. Primer sequences were summarized in Table 1. and pan-AKT (all from CST, Danvers, MA, USA). These primary antibodies were diluted at 1:1000. Horseradish-peroxidase-conjugated goat anti-mouse or anti-rabbit or anti-rat secondary antibodies (1:100,000, Santa Cruz, Dallas, Texas, USA) were used and SuperSignal West Pico chemiluminescent substrate (Thermo Fisher) was applied for protein detection. LabWorks v4.6 software (UVP, Inc., Upland, CA, USA) was applied for quantification of Western blot. The background was subtracted, and the signal of each target was normalized to that of the β-actin band.
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