Total RNA was extracted from liver cancer cell lines by TRIzol reagent (Invitrogen, USA) and reverse transcribed into cDNA using the GoScript™ Reverse Transcription System (Promega, USA). RT-qPCR was then performed using LightCycler® 480 SYBR Green I Master Mix (Roche, USA). For mature miRNAs, cDNA was reverse-transcribed using the Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Japan) and then amplified by LightCycler® 480 SYBR Green I Master Mix (Roche, USA). U6 and GAPDH were used as internal references for miR-297 and mRNAs, respectively. The 2−△△CT method was used to determine relative gene expression. The specific primers for PCR amplification are provided in Supplementary Table 2 .
Lightcycler 480 sybr green 1 master mix
Manufactured by Roche
Sourced in Switzerland, Germany, United States, United Kingdom, France, Canada
The LightCycler 480 SYBR Green I Master Mix is a ready-to-use solution for real-time PCR analysis. It contains SYBR Green I dye and all necessary reagents for amplification and detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (169 mentions)
Trizol reagent, by Thermo Fisher Scientific (137 mentions)
Rneasy mini kit, by Qiagen (114 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (78 mentions)
Trizol, by Thermo Fisher Scientific (72 mentions)
Lightcycler 480 system, by Roche (69 mentions)
Lightcycler 480 2, by Roche (62 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (56 mentions)
Lightcycler 480 real time pcr system, by Roche (48 mentions)
Lightcycler 480 instrument 2, by Roche (37 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (37 mentions)
Iscript cdna synthesis kit, by Bio-Rad (32 mentions)
Lightcycler 480 instrument, by Roche (31 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (30 mentions)
Primescript rt reagent kit, by Takara Bio (30 mentions)
Quantitect reverse transcription kit, by Qiagen (25 mentions)
Rneasy kit, by Qiagen (22 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (21 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (21 mentions)
Superscript 3, by Thermo Fisher Scientific (20 mentions)
926 protocols using lightcycler 480 sybr green 1 master mix
1
Quantifying mRNA and miRNA Expression
2
Quantitative RT-PCR Liver RNA Analysis
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Total liver RNA was isolated from liver tissue using the TRIzol Reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. The isolated RNA samples were converted to cDNA using reverse transcriptase (SuperScript III; Invitrogen) and oligo (dT) primers. All PCR reactions were performed on the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany) using LightCycler480 SYBRGreen I Mastermix (Roche Diagnostics) in standard 10 μL reaction volumes as follows: 4 μL (100 ng) cDNA, 0.5 μL of 10 pmol/L sense primer, 0.5 μL of 10 pmol/L antisense primer, and 5 μL LightCycler 480 SYBRGreen I Mastermix (Roche Diagnostics). To guarantee the reliability of the obtained results, all samples were processed in triplicate and performed using a negative control. The values obtained were normalized to the control and expressed as fold changes.
3
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated using RNAprep Pure Plant kit (TIANGEN, Beijing, China). To avoid degradation all steps were carried out at low temperature. RNA quantity and quality was determined using both Nanodrop2000-C spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and gel electrophoresis. Approximately 1 μg of qualified total RNA of each sample was used to synthesize first strand complementary DNA according to the manufacturer’s instructions of the PrimeScript® RT Reagent kit after gDNA Eraser treatment (TaKaRa, Dalian, China).
Gene-specific primers for qRT-PCR were designed using the NCBI Primer-Blast Tool. Detailed information of all the primers used in this study is listed in Additional file8 : Table S4. qRT-PCR analysis was performed on a LightCycler TM 480 II System (Roche Applied Science, Basel, Switzerland) using LightCycler 480 SYBR green 1 Master mix (Roche). The relative amounts of candidate genes were calculated with the 2-ΔΔCT method. The cotton histone3 (GenBank: AF024716.1/locus_ID of NAU-NBI, v1.1: Gh_D03G0370) gene was used as the reference gene. All qRT-PCRs were performed in triplicate.
Gene-specific primers for qRT-PCR were designed using the NCBI Primer-Blast Tool. Detailed information of all the primers used in this study is listed in Additional file
4
Quantification of Aortic Gene and Protein Expression
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Total mRNA from the whole aorta was extracted using TRIzol reagent, purified with DNase, and reverse transcribed with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, 04897030001).28 The target genes were quantified by real‐time PCR using LightCycler 480 SYBR Green 1 Master Mix and a LightCycler 480 QPCR System (Roche Diagnostics, Indianapolis, IN). The relative transcription levels of target genes were normalized against GAPDH gene expression. Proteins were extracted from the aortas, which were homogenized in lysis buffer as described previously.29 Then, 5 μg of protein was separated by SDS‐PAGE and transferred to polyvinylidene fluoride membranes, which were blocked in Tris‐buffered saline/Tween 20 for 1 hour at room temperate and incubated with primary antibodies overnight at 4°C. Next, the membranes were incubated with secondary antibodies and treated with enhanced chemiluminescence reagents before being visualized using a FluorChem E Imager (ProteinSimple, San Jose, CA). Specific protein expression was normalized against GAPDH or Lamin B protein expression.
5
Quantifying Bacterial 16S rRNA Copy Number
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Real-time quantitative PCR was performed using the LightCycler 480 (Roche, Germany) apparatus. Universal bacterial primers [43, 49 (link)] were used to determine the 16S rRNA copy number. Real-time PCR analyses were performed using LightCycler 480 SYBR Green 1 Master Mix (Roche, Germany) following the manufacturer’s instructions and at an annealing temperature of 56 °C. The DNA concentration was measured in a Qubit Broad Range assay kit (Invitrogen, Thermo Fisher, MA, USA) and the concentration was normalized to 5 ng µl−1. A 10 µl reaction was used with 0.1 mM of the universal primers. DNase-free water was used for negative controls. Standard curves were generated from E. coli http://fluidigm-melting-curve-analysis.software.informer.com/ ).
6
Quantifying Beclin1 mRNA in Mouse Sperm
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Sperm samples of mice were homogenized in TRIzol (Invitrogen, Massachusetts, USA) on ice, and total mRNA was extracted. mRNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche, USA) according to the manufacturer’s instructions. All experiments were performed in triplicate, and quantitative RT-PCR analysis was carried out using the Light Cycler 480 SYBR Green 1 Master Mix (04707516001, Roche, USA). The primer sequences for Beclin1 were: forward: 5′-ATCCTGGACCGTGTCACCATCCAGG-3′; reverse: 5′-GTTGAGCTGAGTGTCCAGCTGG-3′. The primer sequences for Gapdh were: forward: 5′-GTTGTCTCCTGCGACTTCA-3′; reverse: 5′-GGTGGTCCAGGGTTTCTTA-3′. The mRNA levels were analysed using the 2−ΔΔCt method and normalized to Gapdh. The qPCR analysis was performed as described in a previous report79 (link).
7
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from cultured cells, organoids, or flash-frozen tissue using an RNeasy Mini Kit (QIAGEN, 74106). cDNA was prepared using the ProtoScript First Strand cDNA Synthesis Kit (New England BioLabs, E6300). Real-time qRT-PCR was performed on the LightCycler 480 instrument (Roche) using LightCycler 480 SYBR Green 1 MasterMix (Roche, 04887352001) and analyzed using the associated software. Samples were run in triplicate and normalized to Actb as a control. The primer sequences are listed in Supplemental Table 3 .
8
Quantification of mRNA and Protein Levels in Cardiac Fibroblasts
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Total RNA from cultured CFs was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then reverse-transcribed using a Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics) as previously described (29 (link)). The expression levels of target genes were quantified via real-time PCR using a LightCycler 480 SYBR Green 1 Master Mix and a Light-Cycler 480 qPCR system (Roche Diagnostics). The relative transcription levels of the target genes were normalized GAPDH, while the level of miR-144 was normalized to U6 level. The primers for miR-144 (cat. no. HmiRQP0190) were obtained from GeneCopoeia and the other primers are listed in the Table I . Total protein was extracted from the cultured CFs and the concentration of the proteins was determined using a BCA assay. Equal concentrations of proteins were separated via SDS-PAGE (10% gel) and then transferred onto PVDF membranes. After blocking with 5% non-fat dried milk in TBS for 1 h, the membranes were incubated with primary antibodies. Membranes were then incubated with a secondary antibody and treated with enhanced chemiluminescence reagent. Images were captured using a Molecular Imager ChemiDoc™ XRS+ and quantified with Image Lab™ Software (version 5.1). The expression levels of specific proteins were normalized against GAPDH. The associated antibodies are listed in Table II .
9
Gene Expression Analysis in Connective Tissue
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To examine the relative mRNA expression of CollagenIα, Collagen IIIα, connective tissue growth factor (CTGF), TGF-β1, and α-smooth muscle actin (SMA), total RNA was collected using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the cDNA was used as a template for reverse transcription polymerase chain reaction (RT-PCR) amplification and detection of the gene expression level. Quantification RT-PCR was carried out using a one-step qPCR kit (Roche; Basel, Switzerland). PCR amplifications were quantified using a LightCycler 480 SYBR Green 1 Master Mix (Roche). The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used to normalize gene mRNA expression.
10
Quantifying mRNA Levels via RT-PCR
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To measure the levels of mRNA, total RNA was isolated from cultured cells or mouse brain tissue using TRIzol reagent (Invitrogen) and reverse transcribed to complementary DNA using the iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR (RT-PCR) was performed using Light Cycler 480 SYBR Green 1 Master Mix (Roche). The primers are listed in Table 1 . The relative expression levels of target proteins were normalized to that of GAPDH and assessed using the 2−ΔΔCt method.
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