Extracted RNA was reverse-transcribed to generate first-strand cDNA (QuantScript RT Kit, TIANGEN) before qPCR. Quantitative Realtime PCR was performed on DNase-treated RNA using SuperReal PreMix Plus (SYBR Green) (TIANGEN) according to the manufacturer’s directions on a Realtime PCR System (eppendorf).
Real time pcr system
Manufactured by Eppendorf
Sourced in Germany, United States
The Real-time PCR system is a laboratory instrument used for the amplification and detection of specific DNA sequences in real-time. It is designed to measure the accumulation of DNA amplification products during the PCR (Polymerase Chain Reaction) process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Rnaiso plus kit, by Takara Bio (4 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (3 mentions)
Quantscript rt kit, by Tiangen Biotech (2 mentions)
Sybr green premix ex taq, by Takara Bio (2 mentions)
Omniscript reverse transcriptase kit, by Qiagen (2 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Sybr premix ex tag 2, by Takara Bio (2 mentions)
One step primescript mirna cdna synthesis kit, by Takara Bio (2 mentions)
Sybr green master mix, by Takara Bio (2 mentions)
Pcr master mix, by Promega (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Sybr premix ex taqtm 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
37 protocols using real time pcr system
1
Reverse Transcription and qPCR Analysis
2
Analyzing MCP-1 Expression in Macrophages
3
Quantifying Gene Expression in Skin and Cell Lines
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Total RNA was extracted from skin samples or HaCaT cells using Trizol reagent (Invitrogen). Quantitative realtime PCR (qRT-PCR) was performed on a real-time PCR system (Eppendorf, Germany) using SYBR Green Premix Ex Taq (TaKaRa, Dalian, China). Primers were synthesized by Baiaoke Biotech (Beijing, China) and the following primers were used: human GAPDH: forward 5′-TGTTGCCATCAATGACCCCTT-3′, and reverse 5′-CTCCACGACGTACTCAGCG-3′; human C10orf99: forward 5′-GCTTCTCTGCTTCTCCATCTTCT-3′, and reverse 5′-TTCAGGTTTGTTGAGTTGGG-3′; human TNF-α: forward 5′-TCCTTCAGACACCCTCAACC-3′; and reverse 5′-AGGCCCCAGTTTGAATTCTT-3′; human IL-6: forward 5′-AAGCCAGAGCTGTGCAGATGAGTA-3′, and reverse 5′-TGTCCTGCAGCCACTGGTTC-3′; human IL-8: forward 5′-GTCCTTGTTCCACTGTGCCT-3′, and reverse 5′-GCTTCCACATGTCCTCACAA-3′; human BD2: forward 5′- TCAGCCATGAGGGTCTTGTA-3′, and reverse 5′-GGATCGCCTATACCACCAAA-3′; mouse 2610528A11Rik: forward 5′- TTCTAGCCCTTTCCGGTCTG-3′, and reverse 5′- CACCACCCATGACTTGACTG-3′; mouse β-actin: forward 5′-CCTCTATGCCAACACAGTGC-3′, and reverse 5′- ACATCTGCTGGAAGGTGGAC-3′. GAPDH or β-actin was used as internal controls. The relative quantification of gene was obtained using the 2−ΔΔCT method relative to the internal control.
4
qRT-PCR Validation of RNA-Seq Differentially Expressed Genes
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qRT-PCR was used to confirm the accuracy of the differential expression of DEGs via RNA-Seq. Total RNA was isolated from the leaf samples and treated with DNase I to remove any genomic DNA contamination. The single-stranded cDNAs used for real-time PCR analysis were synthesized using a PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). qRT-PCR was carried out using SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China) on an Eppendorf Real-Time PCR System (Mastercycler® ep realplex, Germany) according to the manufacturer's protocol, and the amplification was conducted as described by Ren et al. (2014 (link)). The C. sinensis β-actin gene (Csβ-actin, GenBank: HQ420251.1 ) was amplified as an internal reference standard, and the relative expression levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Three biological and three technical replicates were performed for each sample, and the primers used for qRT-PCR are listed in Supplementary S1 .
5
qRT-PCR Analysis of O. furnacalis Genes
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For qRT-PCR analyses, total RNA was extracted from O. furnacalis larvae and adults using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Ambion, Austin, TX, USA), according to the manufacturer’s instructions. cDNAs were synthesized using the Omniscript Reverse Transcriptase kit (Qiagen, Hilden, Germany) in a 20 μL reaction mixture containing 1 μg total RNA. qRT-PCR analysis for OfMasc and Ofdsx mutants was performed using a SYBR Green Real-Time PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on an Eppendorf Real-Time PCR System. The PCR conditions were as follows: initial incubation at 95 °C for 5 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. O. furnacalis actin was used as an internal control [32 (link)]. The gene-specific primers used for qRT-PCR are listed in Table 2 .
6
Brain Tissue RNA Extraction and qPCR Analysis
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RTq-PCR was performed as described previously (23 (link)). Total RNA was isolated from brain tissues using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse transcribed using HiScript II Reverse Transcriptase (R201-01/02; Vazyme Biotech Co., Ltd., Nanjing, China) and random primers in a PCR reaction procedure (25°C for 5 min, 42°C for 15 min and 85°C for 5 min). qPCR was performed on the Mastercycler ep realplex qPCR System (Eppendorf, Hamburg, Germany) with the SYBR-Green qPCR Master Mix (Vazyme Biotech Co., Ltd., Jiangsu, China) with specific primers for Miz1 and β-actin as an internal control. An Eppendorf Real Time PCR system was used with the following parameters: One cycle at 95°C for 30 sec, followed by 40 cycles at 95°C 5 sec, 60°C for 34 sec, and one cycle at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. The median value of the replicates for each sample was calculated according to the 2−ΔΔCq method (24 (link)). Data presented were the mean from 3 independent experiments. Primers used were as follows: Miz1 forward, 5′-AGGCACACTGTCTGAGAAGAGA-3′ and reverse, 5′-TGGTTCAGCTGCTCCAAGA-3′; and β-actin forward, 5′-ACGGTCAGGTCATCACTATCG-3′ and reverse, 5′-GGCATAGAGGTCTTTACGGATG-3′.
7
Quantitative PCR Analysis of Gene Expression
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Cells were seeded into 6-well plates and received treatments as indicated. Total RNAs was extracted using an RNAiso plus kit (TaKaRa, Japan). Complementary DNA was synthesized through reverse transcription using ReverTra Ace qPCR RT Kit (TOYOBO, Japan). Quantitative PCR analysis of cDNA was performed with SYBR Green reaction master mix on a real-time PCR system (Eppendorf International, Germany). Target mRNA levels were normalized to the level obtained for GAPDH. Changes in transcript level were calculated using DD△Ct method. The primers used in this experiment were listed in Supporting Information Table S1 .
8
Quantitative Gene Expression Analysis
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Total RNAs was extracted using an RNAiso plus kit (TaKaRa). Complementary DNA was synthesized through reverse transcription using ReverTra Ace qPCR RT Kit (TOYOBO). Quantitative PCR analysis of cDNA was performed with SYBRGreen reaction master mix on a Real‐time PCR System (Eppendorf International). Target mRNA levels were normalized to the level obtained for GAPDH. Changes in transcript level were calculated using DD△Ct method. The primers used in this experiment were listed in Table 1 .
9
Quantitative Real-Time PCR Protocol
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Total RNA was extracted from cells using Tripure (Roche) according to the manufacturer's protocol. Reverse transcription was performed using the PrimeScript RT-PCR Kit (TaKaRa) followed by semiquantitative real-time PCR with specific primers described in using SYBR Premix Ex Taq (TaKaRa). Real-time PCR was performed on a real-time PCR system (Eppendorf, Germany) with an initial step of 10 min at 95°C, followed by 40 cycles of 30 s denaturation at 95°C, 30 s annealing at 60°C, and 20 s extension at 72°C. Melting curves were assessed over the range 60-99°C to ensure specific DNA amplification. Target gene expression was normalized to β-actin expression and is shown as levels relative to control samples.
10
Reverse Transcription qPCR Protocol
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cDNAs were synthesized using 1 μg total RNA from each developmental stage as templates by using an Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany) in a 20 μL reaction mixture. The primers used for the RT-qPCR are listed in Table S1 . RT-qPCR was performed on an Eppendorf Real-time PCR System using the following conditions: a 2 min denaturing cycle at 95 °C, 35 cycles of 1 min at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by a final extension at 72 °C for 10 min.
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