To determine the siRNA efficiency, Tca-8113 cells were infected with lentivirus expressing either ZFX-siRNA or scrambled siRNA and cultured for another 48 h. Then, total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. M-MLV reverse transcriptase (Promega) and Oligo dT primers (Sangon, Shanghai) were used for reverse transcription to produce cDNAs. Briefly, a 10 μl mixture containing 2 μg RNA, 0.5 μg Oligo dT primers was incubated at 70 °C for 10 min and then cooled on ice. Then, buffer, reverse transcriptase, RNase inhibitor, and dNTPs were added to a final 20 μl mixture and incubated at 42 °C for 1 h. ZFX expression was quantified with real-time quantitative PCR using a Real-Time PCR machine TP800 (Takara). Here, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers used in real-time PCR were as follows: GAPDH for 5′-TGACTTCAACAGCGACACCCA-3′; GAPDH reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′; ZFX for 5′-GGCAGTCCACAGCAAGAAC-3′; ZFX reverse: 5′-TTGGTATCCGAGAAAGTCAGAAG-3′. Messenger RNA (mRNA) levels of ZFX were normalized against GAPDH, and the comparative CT method was chosen to quantify ZFX expression in Microsoft Excel.
Real time pcr machine tp800
Manufactured by Takara Bio
Sourced in Japan, China
The Takara Bio Real-Time PCR machine TP800 is a compact, high-performance instrument designed for accurate and reliable quantitative analysis of nucleic acids. It utilizes advanced optical detection technology to provide real-time monitoring of PCR amplification. The TP800 is capable of processing up to 96 samples simultaneously, making it a versatile tool for a wide range of applications in molecular biology and genomics research.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Promega (5 mentions)
Sybr master mixture, by Takara Bio (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Oligo dt primer, by Sangon (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Tiangen Biotech (2 mentions)
Oligo dt primers, by RiboBio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
8 protocols using real time pcr machine tp800
1
Quantifying siRNA Knockdown Efficiency
2
C1QTNF6 Gene Expression Quantification
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Total RNA was isolated from cells using TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcription was performed to synthesize cDNAs using M-MLV reverse transcriptase (Promega) and OligodT primers (Sangon, Shanghai). C1QTNF6 mRNA expression was detected by quantitative real-time PCR using SYBR Master Mixture (Takara, Japan) on a Real-Time PCR machine TP800 (Takara). The cycling parameters were: 95 °C for a 30 s hot start followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers used were as follows: GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′, GAPDH reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′, C1QTNF6 forward: 5′-GAAAGGGTCTTTGTGAACCTTGA-3′, and C1QTNF6 reverse: 5′-CTGCGCGTACAGGATGACAG-3′. The relative mRNA expression was calculated using the 2−ΔΔCt method.
3
Quantification of MAT2B mRNA Expression
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Total RNA was extracted and purified using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. RT was performed using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) and Oligo dT primers (Guangzhou RiboBio Co., Ltd., Guangzhou, China) to obtain cDNA for 10 min at 70°C. The cycling parameters were 95°C for a 30 sec hot start followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. MAT2B mRNA expression was measured with RT-qPCR using SYBR master mixture (Takara Biotechnology Co., Ltd., Dalian, China) on a Real-Time PCR machine TP800 (Takara Bio, Inc., Otsu, Japan). Primers used were as follows: GAPDH: forward, 5′-TGACTTCAACAGCGACACCCA-3′, reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′; MAT2B: forward, 5′-ACAGAGAGGAAGACATACCAG-3′, reverse, 5′-GTTCA TTGCCAGACCAGTG-3′. The relative MAT2B expression was normalized to that of GAPDH, and data analysis was conducted using the comparative Cq method. The cycling parameters included an initiation step at 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The primer sequences were obtained from Guangzhou RiboBio Co., Ltd. The relative mRNA expression (MAT2B/GAPDH) was determined using the 2−ΔΔCq method, ΔCq = Cq of target gene - Cq of GAPDH and ΔΔCq = average Cq of negative control group - Cq of each sample (14 (link)).
4
RNA Isolation and Real-Time PCR Analysis
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Trizol Reagents (Invitrogen, Carlsbad, CA, USA) was selected for isolating total RNA from cells of different groups. And templates of cDNA were accomplished via real-time PCR using cDNA M-MLV kit (Promega, Fitchburg, WI, USA). Then the products were used to generate real-time PCR templates. Primer pairs for real-time PCR with SYBR Master Mixture (Takara Biotech, Dalian, China) monitored gene expression. Real-time PCR machine TP800 (Takara Biotech) was responsible for analyzing the results. PCR conditions were strictly controlled as below: ten minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Compared with control, the fold changes of mRNA levels were calculated via the Δ ΔCt method. GAPDH was used as an internal reference for normalization.
5
Quantifying C1QTNF6 mRNA Expression
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Total RNA was isolated from cells using TRIzol (Invitrogen, USA) according to the manufacturer's instructions. Reverse transcription was performed to synthesize cDNAs using M-MLV reverse transcriptase (Promega) and OligodT primers (Sangon, Shanghai). C1QTNF6 mRNA expression was detected with quantitative real-time PCR using SYBR Master Mixture (Takara, Japan) on a Real-Time PCR machine TP800 (Takara). The cycling parameters were 95℃ for a 30s hot start followed by 45 cycles of 95℃ for 5 s and 60℃ for 30s. Primers used here were as follows: GAPDH for:5′-TGACTTCAACAGCGACACCCA -3′, GAPDH reverse:5′-CACCCTGTTGCTGTAGCCAAA -3′, C1QTNF6 for: 5′-GAAAGGGTCTTTGTGAACCTTGA -3′, and C1QTNF6 reverse: 5′-CTGCGCGTACAGGATGACAG -3′. The relative mRNA expression was calculated using the 2 -ΔΔCt method.
6
Poplar RNA Extraction and RT-PCR Analysis
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Total RNA was extracted from different tissues of poplar plants using the Trizol Reagent (Tiangen, China). The gene-specific primers are listed in Supplementary Table S1 . Semi-quantitative reverse transcription (RT-PCR) conditions were an initial denaturation step at 94 °C for 3 min, 28 cycles of 94 °C for 30 s, 58 °C, and 72 °C for 1 min, and an extension step at 72 °C for 10 min. The amplification products were resolved by 1% (w/v) agarose gel electrophoresis and visualized with ethidium bromide under UV light. A TP800 Real-Time PCR machine (TaKaRa, Japan) was used for quantitative real-time reverse transcription-PCR (qRT-PCR) analysis. The poplar Ptr18S gene was used as internal references to normalize the expression data. Three biological and three technical replicates were performed for each gene.
7
Quantitative RT-qPCR Analysis of Gene Expression
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Plant materials such as roots, stems, xylem, bast, old and young leaves in good growth condition were selected and ground thoroughly using liquid nitrogen, and then total RNA was extracted using the Trizol Reagent (Tiangen, China), followed by reverse transcription of cDNA using the PrimeScript™ RT reagent Kit (Takara, Shiga, Japan). Specific quantitative primers were designed as needed, and the quantitative RT-qPCR was performed using SYBR Premix ExTaq™ (Takara) in a TP800 Real-Time PCR machine (Takara). The UBQ11 gene was used as an internal control in poplar and AtUBC in Arabidopsis. The RT-qPCR reaction conditions were 95°C, 30 s; 40 cycles: 95°C, 5 s; 60°C, 1 min; 95°C, 15 s; 60°C, 30 s; 95°C, 15 s. cycles: 95°C, 5 s; 60°C, 1 min; 95°C, 15 s; 60°C, 30 s; 95°C, 15 s. Three biological replicates were required for each gene of the RT-qPCR, and the primers involved were listed in Table S1 Figure 1F
8
Quantitative Analysis of ATP1B3 mRNA Expression
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Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, California, USA) and was synthesized into cDNA using M-MLV reverse transcriptase (Promega, Wisconsin, USA) according to the manufacturer's instructions. ATP1B3 mRNA expression was measured by real-time quantitative PCR using SYBR QPCR mastermix (Vazyme, Nanjing, China) in a TP800 Real-Time PCR machine (TaKaRa). The primers used were as follows: GAPDH forward,5’-TGAC TTCAACAGCCACACCCA-3’, GAPDH reverse, 5’-CAC CCTGTTGCTGTAGCCAAA -3’; ATP1B3 forward, 5’-AA CCCGACCACCGGAGAAAT-3’, and ATP1B3 reverse, 5’-TGAGAGTCTGAAGCATAACCCA-3’. GAPDH was used as an internal control for both reverse-transcription PCR and real-time quantitative PCR. PCR was performed with the following cycle profile: 94°C for 5 min; 94°C for 30 s, 53°C for 30 s, and 72°Cfor 35 s for 35 cycles, with a final extension step at 72°C for 2 min. The reverse-transcription PCR products were analysed by electrophoresis on a 2.0% agarose gel to compare the ATP1B3 mRNA levels among different cell lines. The relative mRNA expression was determined using the 2-ΔΔCt method to calculate the ATP1B3 gene silencing efficiency. All samples were assayed in triplicate.
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