The total RNA was isolated from cells and tissues using Trizol reagent (Life Technologies). First‐strand cDNA was carried out using reverse transcription reagents (ABI). Quantitative real‐time PCR (qRT‐PCR) was performed using SYBRH Select Master Mix for CFX (Invitrogen) and using the CFX Connet TM real‐time PCR system (Bio‐Rad). GAPDH and U6 were chosen as the internal standard. The primer sequences used are shown in Table 1 .
Cfx connet tm real time pcr system
Manufactured by Bio-Rad
Sourced in United States
The CFX Connect™ real-time PCR system is a compact and versatile instrument designed for accurate and reliable quantitative real-time PCR analysis. It features a high-performance optical detection system, thermal cycling capabilities, and user-friendly software for efficient nucleic acid quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Sybrh select master mix for cfx, by Thermo Fisher Scientific (4 mentions)
Reverse transcription reagent, by Thermo Fisher Scientific (3 mentions)
Taqmanhmicrorna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Mirna cdna kit, by CWBIO (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Ultrasybr mixture, by CWBIO (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using cfx connet tm real time pcr system
1
Quantitative Gene Expression Analysis
2
qRT-PCR Analysis of Gene Expression
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Total RNAs from cell lines were extracted with TRIzol reagent (Invitrogen, CA) and the first-strand cDNA was generated using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) or miRNA cDNA Kit (CWBio, China) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using UltraSYBR mixture (Cwbio, China) and conducted using the CFX Connet TM real-time PCR system (Bio-Rad, USA). The quantification analysis was analyzed by the 2-ΔΔCT method [26 (link)]. GAPDH and U6 mRNA were used for normalization. All the primers are listed in Table 1 .
3
Comprehensive RNA Extraction and qPCR Analysis
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Total RNA was extracted from clinical samples and cells using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RNA purity was was examined using spectrophotometry and 100 ng RNA was reserved for complementary DNA(cDNA) synthesis using reverse transcription kit (ABI, CA) or TaqManH MicroRNA reverse transcription kit (ABI, CA) following the manufacturer's protocol. Real-time quantitative PCR was performed using SYBRH select master mix for CFX (Invitrogen, Carlsbad, CA) and the CFX Connet TM real-time PCR system (Bio-Rad, USA). The expression of miR-543 and DKK1 mRNA was normalized to U6 and β-actin respectively. The relative amount of miRNA or mRNA was calculated via the 2-∆∆Ct method and the primer sequences used are shown in Table 2 .
4
Quantitative RT-PCR of RNA Transcripts
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Total RNA was isolated from tumor specimens and cancer cell lines using Trizol reagent (Invitrogen, USA). The purity of RNA was examined by spectrophotometry and the first strand cDNA was synthesized using reverse transcription Reagents (ABI, CA) or the TaqManH MicroRNA Reverse Transcription Kit (ABI, CA) following the manufacturer’s instructions. QRT-PCR was performed using SYBRH Select Master Mix for CFX (Invitrogen) and using the CFX Connet TM real-time PCR system (Bio-Rad, USA). All results were normalized to the expression of GAPDH or snRNA U6. The quantitative analysis was calculated by using 2-ΔΔCt method. All the primers are shown in Table 2 .
5
Quantitative RNA Expression Analysis
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Total RNA was isolated from cancer cell lines using TRIzol reagent (Invitrogen, USA). The purity of RNA was examined by spectrophotometry and the first strand cDNA was synthesized using reverse transcription Reagents (ABI, CA) or the TaqManH MicroRNA Reverse Transcription Kit (ABI, CA) following the manufacturer’s instructions. QRT-PCR was performed using SYBRH Select Master Mix for CFX (Invitrogen) and using the CFX Connet TM real-time PCR system (Bio-Rad, USA). All results were normalized to the expression of GAPDH or snRNA U6. The quantitative analysis was calculated by using 2−ΔΔCt method. All the primers are shown in Table 1
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