The extracted total RNA from mouse aortas and livers (n = 10 per group) in bulk RNA‐seq was also used for qPCR. mRNA was reversely transcribed to cDNA using an Evo M‐MLV RT Master Mix (Accurate Biotechnology, Hunan, China). qPCR was performed using a 5× Priescript RT Master Mix and an SYBR Green Realtime PCR Master Mix (Takara, Shiga, Japan) with an Applied Biosystems ViiA 7 Real‐Time PCR (Thermo Fisher Scientific). The 2−ΔΔCT method[104 ] was adopted to calculate the tested genes which were normalized by β‐actin. The sequence of mRNA primers (synthesized by Tsingke) used in this study was presented in Table S7 of the Supporting Information.
Applied biosystems viia 7 real time pcr
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The Applied Biosystems ViiA 7 Real-Time PCR System is a high-performance, reliable, and flexible real-time PCR instrument. It is designed to enable efficient and accurate gene expression analysis, genotyping, and other real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Maxwell rsc mirna tissue kit mirna, by Promega (2 mentions)
Evo m mlv rt master mix, by Accurate Biology (1 mentions)
Sybr green real time pcr master mix, by Takara Bio (1 mentions)
Maxwell rsc mirna plasma and serum kit, by Promega (1 mentions)
Taq man microrna reverse transcription kit, by Promega (1 mentions)
6 protocols using applied biosystems viia 7 real time pcr
1
Quantitative PCR Analysis of Gene Expression in Mouse Tissues
2
RT-qPCR Gene Expression Analysis
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RT-qPCRs were performed using Applied Biosystems ViiA™ 7 Real-Time PCR (Thermo Fisher). The reaction mixture for RT-qPCR comprised a total volume of 20 μL consisting of the following: 10 μL of 2 × Perfect StartTM Green RT-qPCR SuperMix + DyeII, 0.4 μL each of F/R (Forward and Reverse primers), 1 μL of cDNA template, and 8.2 μL of sterile, double-distilled water. The cycling program comprised an initial denaturation of 10 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing for 30 s at 58 °C, and extension for 32 s at 72 °C. After the reaction, a melting curve analysis from 60 °C to 95 °C was applied to all reactions to ensure consistency and specificity of the amplified products.
3
Quantitative RT-PCR Analysis of Ovarian Tumors
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RNA was extracted from snap frozen ovarian tumor sections, ascites-derived epithelial and mesenchymal cells and ovarian cancer cell lines stored in TRIzol® reagent (Ambion-Life Technologies, CA, USA) by the chloroform: phenol method as described previously (40 (link)). Five hundred ng of total RNA was reverse transcribed using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA), and both relative and absolute qRT-PCR amplification was performed using the Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, NSW, Australia) as described previously (40 (link)–42 (link)). Briefly, for absolute qRT-PCR method, samples were analysed against a known absolute quantity of the gene (fg), and these results were then standardized against an absolute quantity of 18S (fg) and this method was used for comparing tissue samples of different origin. While relative PCR compared Ct values relative to 18S Ct values (δΔCt) and was only used for samples within the same origin. All PCR reactions were performed in triplicate. The sequences and accession numbers of genes analyzed and primers used are listed in Supplementary Table 3
4
Quantifying miRNA and mRNA Levels
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RNA from cell lines and EVs was obtained by automated Maxwell RSC-Promega extractor, using, respectively, the Maxwell RSC miRNA Tissue Kit miRNA (CAT # AS1460, Promega) and Maxwell RSC miRNA Plasma and Serum kit (CAT N.AS1680, Promega).
Retro-transcription for gene detection was performed using High-Capacity cDNA Reverse Transcription Kit. For miRNA, a miRNA-specific RT was carried out using TaqMan™ MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of hsa-miR-200a (Assay ID:001011), hsa-miR-200c (Assay ID: 000505), of hsa-miR-9 (Assay ID: 000583), hsa-miR-222 (Assay ID: 002276), has-miR-221 (Assay ID: 000524) and U6 snRNA (Assay ID: 001973). TaqMan gene expression assays (Cat. N. 4331182, Thermo Fisher Scientific) were used to assess mRNA expression of HMGB1 (Assay ID: Hs01590761g1) and ACTB (Assay ID: Hs01060665_g1). U6 and ACTB were used as normalization controls.
QPCR was performed on cDNA using Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of miRNAs and mRNAs were calculated and quantified using the 2^ΔΔCq method after normalization for the expression of the controls. All procedures were performed according to the manufacturer’s instructions.
Retro-transcription for gene detection was performed using High-Capacity cDNA Reverse Transcription Kit. For miRNA, a miRNA-specific RT was carried out using TaqMan™ MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of hsa-miR-200a (Assay ID:001011), hsa-miR-200c (Assay ID: 000505), of hsa-miR-9 (Assay ID: 000583), hsa-miR-222 (Assay ID: 002276), has-miR-221 (Assay ID: 000524) and U6 snRNA (Assay ID: 001973). TaqMan gene expression assays (Cat. N. 4331182, Thermo Fisher Scientific) were used to assess mRNA expression of HMGB1 (Assay ID: Hs01590761g1) and ACTB (Assay ID: Hs01060665_g1). U6 and ACTB were used as normalization controls.
QPCR was performed on cDNA using Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of miRNAs and mRNAs were calculated and quantified using the 2^ΔΔCq method after normalization for the expression of the controls. All procedures were performed according to the manufacturer’s instructions.
5
Profiling Viral-Induced miRNA Changes
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HHV-6A or mock-infected BCPAP RNA were obtained by an automated Maxwell RSC-Promega extractor, using the Maxwell RSC miRNA Tissue Kit miRNA (CAT # AS1460, Promega, Sydney, Australia).
Retro-transcription was performed using a Taq-Man MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of has-miR-9 (assay ID: 000583), hsa-miR-221 (assay ID: 000524), hsa-miR-222 (assay ID: 002276), hsa-miR-146a-5p (assay ID: 000468), hsa-miR-155-5p (assay ID: 002623), and U6 snRNA (assay ID: 001973).
QPCR was performed using an Applied Biosystems Vii A 7 Real-Time PCR (Thermo Fisher Scientific). The relative expression levels of miRNAs were quantified using the 2∆∆Cq method after normalization for U6 expression, used as housekeeping. All procedures were performed according to the manufacturer’s instructions.
Retro-transcription was performed using a Taq-Man MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of has-miR-9 (assay ID: 000583), hsa-miR-221 (assay ID: 000524), hsa-miR-222 (assay ID: 002276), hsa-miR-146a-5p (assay ID: 000468), hsa-miR-155-5p (assay ID: 002623), and U6 snRNA (assay ID: 001973).
QPCR was performed using an Applied Biosystems Vii A 7 Real-Time PCR (Thermo Fisher Scientific). The relative expression levels of miRNAs were quantified using the 2∆∆Cq method after normalization for U6 expression, used as housekeeping. All procedures were performed according to the manufacturer’s instructions.
6
RNA Extraction and qRT-PCR Analysis
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RNA was extracted from RNAi-treated Cont, T2-KD, CRISPR/Cas9 TIMP-2 edited gRNA1, gRNA2, CRISPR control and parental OVCAR5 cells using TRIzol® reagent (Ambion-Life Technologies, Carlsbad, CA, USA) followed by the chloroform: phenol method as described previously [16 (link)]. Five hundred ng of total RNA was reverse transcribed using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) and qRT-PCR amplification was performed using the Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, NSW, Australia) as described previously [16 (link), 26 (link)]. Additional file 1 : Table S1 lists the sequences and accession numbers of genes analysed. Data are presented as relative expression normalized to housekeeping gene 18S. The experiments were repeated three times in triplicate.
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