Total RNA was isolated from the tissues using an RNeasy Total RNA Isolation Kit (TaKaRa Bio Inc., Japan) and reverse-transcribed into cDNA (TaKaRa Bio Inc., Japan). Then, SYBR Green (DBI, Germany) quantitative PCR analysis reactions were performed using the Roche LightCycler 480 Detection System (Roche, Belgium). Each reaction comprised 10 μl of SYBR Green, 1 μl of cDNA, 1 μl of each primer pair (10 μmol/μl), and 8 μl of distilled water. The beta-actin gene was simultaneously detected as an endogenous reference. The relative gene expression levels were quantified using the 2−ΔΔt method, and the results are expressed as the fold change relative to the endogenous reference. The primer sequences are listed in Table 2 .
Lightcycler 480 detection system
Manufactured by Roche
Sourced in Switzerland, United States, Germany, France, China, United Kingdom, Japan, Belgium
The LightCycler 480 detection system is a real-time PCR instrument designed for high-throughput gene expression analysis and genotyping. It features a 96-well or 384-well microplate format and supports a variety of fluorescence detection chemistries for flexible experimental design. The system provides precise temperature control and optical detection capabilities to enable accurate and reproducible quantitative PCR results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (39 mentions)
Rneasy mini kit, by Qiagen (23 mentions)
Quantitect reverse transcription kit, by Qiagen (9 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (8 mentions)
Primescript rt master mix, by Takara Bio (7 mentions)
Primescript rt reagent kit, by Takara Bio (6 mentions)
Rneasy kit, by Qiagen (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Oligo dt primer, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Cdna synthesis kit, by Takara Bio (5 mentions)
Tripure isolation reagent, by Roche (5 mentions)
Sybr green master mix, by Roche (5 mentions)
Sybr green 1 master, by Roche (5 mentions)
Transstart green qpcr supermix, by Transgene (4 mentions)
Sybr pcr master mix, by Roche (4 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (4 mentions)
Qpcrbio sygreen lo rox supermix, by PCR Biosystems (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
167 protocols using lightcycler 480 detection system
1
Quantitative PCR Analysis of Gene Expression
2
Liver Gene Expression Analysis by RT-PCR
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For analysis of gene expression in the livers, ∼15 mg of frozen liver tissue was homogenized with TRIzol reagent (Ambion, Cat No. 15596-018) and RNA was extracted following the manufacturer’s protocol. The concentration of extracted RNA was determined using Nanodrop ND-1000 spectrophotometer (Thermo Scientific). First-strand cDNA was synthesized from 2.0 μg of total RNA with Superscript III first-strand synthesis kit (Invitrogen) according to the manufacturer's protocol. For real-time RT-PCR reactions, the cDNA was diluted 15-fold. Sequences of the primers are available by request. Real-time PCR analysis was conducted on Roche LightCycler480 detection system (Roche Diagnostics) with SYBR Green as probe (LightCycler480 CYBR Green I Master, Roche). Relative gene expression levels were calculated using the comparative Ct method by normalization to geometric mean of expression levels of four housekeeping genes (GAPDH, β-actin, m18S, and Hprt1). Unpaired t-test was used to test for statistical significance.
3
RNA Isolation and RT-qPCR Analysis
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Total RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Toral RNA was reverse transcribed into cDNA using Super M-MLV Reverse Transcriptase and 2×Power Taq PCR MasterMix (Bioteke Corporation), according to the manufacturer's protocol. Briefly, for RT, 1 µl oligo (dT)15 and 2 µl dNTPs (2.5 mM each) were added, and then ddH2O was added to a total volume of 14.5 µl. The following thermocycling conditions were used for reverse transcription: 10 min at 25°C, 50 min at 42°C and 5 min at 95°C. The both forward and reverse primers for MEG3, TP53 and ACTB were provided by Shanghai GenePharma Co., Ltd. (Table I ).
qPCR was performed using the following thermocycling conditions: Initial denaturation at 95°C for 3 min; 40 cycles of amplification at 95°C for 12 sec and 60°C for 40 sec; and final extension (72°C for 5 min). qPCR was performed using the SYBR Green qPCR Detection kit (Tiangen Biotech Co., Ltd.) and the Roche LightCycler 480 Detection system (Roche Diagnostics). Relative mRNA expression levels of MEG3 and TP53 were normalized to the internal reference gene ACTB using the 2−ΔΔCq method (23 (link)). RT-qPCR was performed in triplicate.
qPCR was performed using the following thermocycling conditions: Initial denaturation at 95°C for 3 min; 40 cycles of amplification at 95°C for 12 sec and 60°C for 40 sec; and final extension (72°C for 5 min). qPCR was performed using the SYBR Green qPCR Detection kit (Tiangen Biotech Co., Ltd.) and the Roche LightCycler 480 Detection system (Roche Diagnostics). Relative mRNA expression levels of MEG3 and TP53 were normalized to the internal reference gene ACTB using the 2−ΔΔCq method (23 (link)). RT-qPCR was performed in triplicate.
4
Quantification of LINC00473 and PEBP1 Expression
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Total RNA was extracted from the cultured cells using a RNAiso plus kit (Takara Bio Inc.), and was then reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara Bio Inc.). qPCR was performed using a SYBR Premix Ex Taq II kit (Takara Bio Inc.) along with a Roche LightCycler 480 Detection System (Roche Diagnostics) as instructed by the manufacturer. The RT-qPCR thermocycling conditions were as follows: Denaturation at 95°C for 30 sec; 40 cycles at 95°C for 5 sec and 60°C for 30 sec; dissociation at 95°C for 5 sec and 60°C for 1 min; and cooling at 50°C for 30 sec. The primers used in the present study were provided by Guangzhou RiboBio Co., Ltd. The sequences of the primers were as follows: Human LINC00473 forward, 5′-GGC AGC CTC AGG TTA CAA AT-3′ and reverse, 5′-AGG AGC AGG TAG GGA AAT GA-3′; human PEBP1 forward, 5′-CTC GCG ATG CTG GTG TAC C-3′ and reverse, 5′-GGA TCC CTG CTT CCC ACA CAG C-3′; and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-CCC ACG CCT CCT CCG TTG AC-3′ and reverse, 5′-ATA CCT GGA AAT CAG CTT TAC AA-3′. The relative expression levels of LINC00473 and PEBP1 were evaluated using the 2−ΔΔCq method (32 (link)), and were normalized to the GAPDH levels. The experiment was performed in triplicate.
5
Quantitative Analysis of Apoptosis Genes
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Total RNA was extracted from each group using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. DNase I was used to eliminate genomic DNA, the contaminant. The concentrations of isolated RNA samples were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using a M-MuLV First-Strand cDNA Synthesis Kit (Sangon Company, Shanghai, China) according to the manufacturer’s instructions. The total reaction volume was 20 μL, which included 1 μg of total RNA, 10 ng/μL of random primer, and RNase-free distilled water.
qRT-PCR was performed using 2X SG Fast qPCR Master Mix (Sangon Company, Shanghai, China). The qRT-PCR instrument was equipped with a Roche LightCycler 480 detection system (Roche Diagnostics, Germany) according to the manufacturer’s instructions. Gene primer sequences are as follows: Bax, sense 5′-AGACACCTGAGCTGACCTTGGAG-3′ and anti-sense 5′-GTTGAAGTTGCCATCAGCAAACA-3′. Bcl-2, sense 5′-TGAACCGGCATCTGCACAC-3′ and anti-sense 5′-CGTCTTCAGAGACAGCCAGGAG-3′. GAPDH, sense 5′-GGCACAGTCAAGGCTGAGAATG-3′ and anti-sense 5′-ATGGTGGTGAAGACGCCAGTA-3′. The PCR reaction of each sample was repeated with 3 holes. Then, the mean threshold cycle was calculated. The results of fluorescence quantitative analysis were calculated by 2-ΔΔCt, using the relative quantification (RQ) value statistics.
qRT-PCR was performed using 2X SG Fast qPCR Master Mix (Sangon Company, Shanghai, China). The qRT-PCR instrument was equipped with a Roche LightCycler 480 detection system (Roche Diagnostics, Germany) according to the manufacturer’s instructions. Gene primer sequences are as follows: Bax, sense 5′-AGACACCTGAGCTGACCTTGGAG-3′ and anti-sense 5′-GTTGAAGTTGCCATCAGCAAACA-3′. Bcl-2, sense 5′-TGAACCGGCATCTGCACAC-3′ and anti-sense 5′-CGTCTTCAGAGACAGCCAGGAG-3′. GAPDH, sense 5′-GGCACAGTCAAGGCTGAGAATG-3′ and anti-sense 5′-ATGGTGGTGAAGACGCCAGTA-3′. The PCR reaction of each sample was repeated with 3 holes. Then, the mean threshold cycle was calculated. The results of fluorescence quantitative analysis were calculated by 2-ΔΔCt, using the relative quantification (RQ) value statistics.
6
Quantification of G-CSF mRNA Expression
7
Quantifying Gene Expression in Skin Tissue
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Approximately 100 mg of frozen dorsal skin tissue of WT and DKO mice samples was homogenized in PURzol reagent (Bio-Rad, catalog no. 7326890) and RNA isolated via the Aurum™ Total RNA Fatty and Fibrous Tissue Pack (Bio-Rad, catalog no. 732-6870). The concentration of isolated RNA was determined with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). ProtoScript® II First Strand cDNA Synthesis Kit (New England BioLabs, catalog no. E6560) was used for cDNA strand synthesis from 1 µg sample RNA. One WT mouse sample was used for several extra reactions simultaneously as a standard for quantification curves. All qPCR was performed with a 15X dilution of cDNA, unless otherwise noted. Curves were calculated with 3X, 9X, 27X, and 81X cDNA dilutions. Real-time PCR analysis was conducted on a Roche LightCycler®480 detection system (Roche Applied Science) with SYBR Green as the probe (LightCycler®480 CYBR Green I Master, Roche Applied Science). Gene expressions were normalized to Gapdh and analyzed as a relative expression of fold-difference from the expression level of WT mice at PD26 of the same sex, using the comparative Ct method. Sequences of primers are available by request.
8
Quantitative RT-qPCR Analysis of Gene Expression
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For each experiment per treatment, RNA was extracted from 25 oocytes using RNeasy Mini Kit (74104, Qiagen) and reverse transcribed into cDNA using Hiscript II Q RT Supermix (R223, Vazyme, China). universal SYBR Green fast qPCR Mix (RK21203, ABclonal, China) was used for real-time fluorescence quantitative detection with Roche LightCycler 480 detection system (Roche Applied Science). PCR primers were listed in Supplementary Table S1 . PCR was performed in a 10 μl reaction system (1 μl cDNA template; 5 μl SYBR Green Mix; 0.25 μl 10 μM forward primer and 0.25 μl 10 μM reverse primer; 3.5 μl ddH2O) using the following amplification condition: pre-denaturation at 95°C for 3 min, 45 cycles of denaturing at 95°C for 5 s and annealing at 60°C for 34 s. Each experiment was performed with three replicates and the cycle threshold (Ct) value was set between 20 and 30. Gapdh was used as the internal control. The gene expression level relative to Gapdh was calculated by the 2−△△Ct method (Livak and Schmittgen, 2001 (link)).
9
Housekeeping Gene Expression Analysis
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The primers of 12 putative housekeeping genes were selected based on previous studies (23, 24) and synthesized by Sangon Company (Shanghai, China) (Table 1). RT-qPCR analysis using 2×SG Fast qPCR Master Mix (Sangon Company, China) was performed on Roche LightCycler 480 detection system (Roche Diagnostics, Germany) as previously described (8, 9) . The RT-qPCR was repeated three times for each sample. The cycle threshold value (Cp value) data were analyzed using the equation of relative quantities (Q): Q = 2 -ΔCp (25) .
10
Quantification of Gene Expression by RT-qPCR
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Total RNA was extracted with TRIzol (#15596018, Invitrogen) and reverse-transcribed to complementary DNA by PrimeScript® RT reagent Kit (Takana, Dalian) using Super Array PCR master mix (SuperArray Bioscience, Frederick, Maryland) as described previously 7 (link), 8 (link), 18 (link), 19 (link). Real-time PCR was run with the double-stranded DNA dye SYBR Green PCR Mastermix in Takana SYBR® Premix Ex TaqTM Kit (Takana, Dalian) following the manufacturer's instruction. Real-time monitoring of PCR amplification was performed using the LightCycler® 480 detection system (Roche). Data were expressed as relative mRNA levels normalized to GAPDH expression level in each sample and represented as mean ± s.d. of at least three independent experiments. The primer sequences are listed as below:
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