RNA was isolated from the mouse RPE following the methodology previously described.26 (link) Total RNA was purified from ARPE-19 cells using the RNeasy Mini Kit (QIAGEN Sciences, Inc., Germantown, MD, USA) following the manufacturer's instructions. Between 100 and 500 ng of total RNA was used for reverse transcription using the Invitrogen SuperScript III First-Strand Synthesis system (Thermo Fisher Scientific, Waltham, MA, USA). The PNPLA2 transcript levels in ARPE-19 cells determined by quantitative RT-PCR were normalized using the QIAGEN QuantiTect SYBR Green PCR Kit in the QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The primer sequences used in this study are listed in Table 1 . Murine PNPLA2 mRNA levels relative to HPRT transcript levels were measured using the QuantStudio 7 Flex Real-Time PCR System using TaqMan gene expression assays (PNPLA2, Mm00503040_m1; HPRT, Mm00446968_m1; Thermo Fisher Scientific). PNPLA2 relative expression to HPRT was calculated using the comparative ΔΔCT method.27 (link)
Quantstudio 7 flex real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan, United Kingdom, Italy, Canada, Singapore, Switzerland, Australia
The QuantStudio 7 Flex Real-Time PCR System is a thermal cycler designed for high-performance real-time PCR applications. It offers a flexible platform with up to 4 thermal blocks, each supporting 96-well plates or 384-well plates. The system features an optical detection module that can measure fluorescence from multiple dyes simultaneously.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (112 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (110 mentions)
Trizol reagent, by Thermo Fisher Scientific (104 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (44 mentions)
Rneasy plus mini kit, by Qiagen (36 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (36 mentions)
Trizol, by Thermo Fisher Scientific (35 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (33 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (29 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (27 mentions)
Quantitect reverse transcription kit, by Qiagen (26 mentions)
Primescript rt master mix, by Takara Bio (25 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (25 mentions)
Taqman probe, by Thermo Fisher Scientific (24 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (21 mentions)
Primescript rt reagent kit, by Takara Bio (20 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (20 mentions)
Rneasy kit, by Qiagen (18 mentions)
Sybr green, by Thermo Fisher Scientific (17 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (16 mentions)
1 063 protocols using quantstudio 7 flex real time pcr system
1
Quantifying PNPLA2 Expression in ARPE-19 Cells
2
Gene Expression Profiling by Real-Time PCR
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Total RNA was extracted, and cDNA was synthesized by reverse transcription as previously described [31 (link)]. The expression of selected genes was quantified by real-time PCR using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific). Gene-specific primers were designed using Primer Express version 2.0 software (Applied Biosystems) and are as follows: 5′-ACCTATGACCTGCTTGGTGC-3′ (HIF-1α forward) and 5′-GGCTGTGTCGACTGAGGAAA-3′ (HIF-1α reverse); 5′-TTAAAGCCCGCCTGACAGA-3′ (IL-1β forward) and 5′-GCGAATGACAGAGGGTTTCTTAG-3′ (IL-1β reverse); 5′-TTACAGAGGGAAAACGACACCT-3′ (GPER forward) and 5′-GTGGGTCTTCCTCAGAAGGG-3′ (GPER reverse); 5′-AGTCCCTGAGCATCTACGGT-3′ (COX2 forward) and 5′-CATCATCAGACCAGGCACCA-3′ (COX2 reverse); 5′-AAGCCACCCCACTTCTCTCTAA-3′ (ACTB forward) and 5′-CACCTCCCCTGTGTGGACTT-3′ (ACTB reverse). Assays were performed in triplicate and the results were normalized for actin beta (ACTB) expression and then calculated as fold induction of RNA expression.
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific).
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific).
3
Quantitative RT-PCR Protocol for Tumor Metastasis
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Total RNA was extracted, and cDNA was synthesized by reverse transcription as previously described [29 (link)]. The expression of selected genes was quantified by real-time PCR using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Milan, Italy). Gene-specific primers were designed using Primer Express version 2.0 software (Applied Biosystems) and are as follows: 5′-CGAGCCCTTTGATGACTTCCT-3′ (c-Fos forward) and 5′-GGAGCGGGCTGTCTCAGA-3′ (c-Fos reverse); 5′-AGCTGTGCATCTACACCGAC-3′ (cyclin D1 forward) and 5′-GAAATCGTGCGGGGTCATTG-3′ (cyclin D1 reverse); 5′-AAGCCACCCCACTTCTCTCTAA-3′ (ACTB forward) and 5′-CACCTCCCCTGTGTGGACTT-3′ (ACTB reverse); 5′-CCTTGGAGCCAAATTTAAAACCT-3′ (CXCR4 forward) and 5′-GCTGGACCCTCTGCTCACA-3′ (CXCR4 reverse); 5′-CAGATGCCCATGCCGATTCT-3′ (CXCL12 forward) and 5′-TTCTTCAGCCGGGCTACAAT-3′ (CXCL12 reverse). Assays were performed in triplicate and the results were normalized for actin beta (ACTB) expression and then calculated as fold induction of RNA expression.
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific, Milan, Italy) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Milan, Italy).
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific, Milan, Italy) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Milan, Italy).
4
Quantitative Evaluation of SARS-CoV-2 Target Genes
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Total RNA was extracted with GenElute, purified with the Total RNA Purification Kit (Sigma Aldrich), and reverse-transcribed to cDNA with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA). Quantitative real-time PCR (qRT-PCR) reactions were prepared using FastStart Universal SYBR Green Master (ROX, Roche) and carried out using QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Data were collected using QuantStudio 7 Flex Real-Time PCR System software version 1.2 (Applied Biosystems) and analyzed using Microsoft Excel 2016 (Microsoft, Redmond, Washington). Quantitative gene expression data were normalized to GAPDH expression. The following primers were used: human ACE2: forward 5′-TGAAGTTGAAAAGGCCATCAG-3′ and reverse 5′-GAGGTCCAAGTGTTGGCTGT-3′; human TMPRSS2: forward 5′-GAGAAAGGGAAGACCTCAGAAG-3′ and reverse 5′-GGTGTGATCAGGTTGTCATAGA-3′; human GAPDH: forward 5′-CCTGCACCACCAACTGCTTA-3′ and reverse 5′-GGCCATCCACAGTCTTCTGAG-3′; SARS-CoV-2 N1 nucleocapsid gene: forward 5′-GACCCCAAAATCAGCGAAAT-3′ and reverse 5′-TCTGGTTACTGCCAGTTGAATCTG-3′. SARS-CoV-2 sgRNA: forward 5′-GTAACAAACCAACCAACTTTCG-3′ and reverse 5′-CATTGTTCACTGTACACTCGATC-3′.
5
Gene Expression Profiling via qRT-PCR
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Total RNA was extracted from TRMs with miRCURY RNA Isolation Kit—Cell and Plant (Exiqon). The RNA was reverse-transcribed to cDNA with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA). Quantitative real-time PCR (qRT-PCR) reactions were prepared using FastStart Universal SYBR Green Master (ROX, Roche) and carried out using QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Data were collected using QuantStudio 7 Flex Real-Time PCR System software version 1.2 (Applied Biosystems) and analyzed using Microsoft Excel 2016 (Microsoft, Redmond, Washington). The full list of primers used can be found in Supplementary Table 2 . Samples were normalized to GAPDH expression.
6
Quantifying Gene and miRNA Expression
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RNA was isolated from cell samples using NucleoSpin RNA Plus kit (Macherey-Nagel) and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative PCR was performed on 1 ng cDNA, in 384-well plates using the SYBR Green PCR Master Mix (Applied Biosystems) with the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). RNA levels were normalized against human GAPDH. Relative amounts of transcripts were determined using the ΔΔ − Ct method and human GAPDH transcript level was used as an internal control for each cell line sample.
miRNA was isolated using mirVana miRNA isolation kit (Ambion; Life Technologies). Reverse transcription was performed with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems) with the Megaplex RT Primers Pool A v2.1 (Applied Biosystems). Quantitative PCR was performed on 1 ng cDNA, in 384-well plates using the TaqMan Gene Expression Master Mix (Applied Biosystems) with the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Relative amounts of transcripts were determined using the Δ Δ − Ct method and human RNU6B was used as an internal control for each cell line sample. The primers were used are described in Table S3.
miRNA was isolated using mirVana miRNA isolation kit (Ambion; Life Technologies). Reverse transcription was performed with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems) with the Megaplex RT Primers Pool A v2.1 (Applied Biosystems). Quantitative PCR was performed on 1 ng cDNA, in 384-well plates using the TaqMan Gene Expression Master Mix (Applied Biosystems) with the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Relative amounts of transcripts were determined using the Δ Δ − Ct method and human RNU6B was used as an internal control for each cell line sample. The primers were used are described in Table S3.
7
Quantitative Analysis of Gene Expression in Stomach and Intestinal Tissues
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Stomach and intestinal tissue from the NC, AM and H-CP groups were homogenized in 1 ml TRIzol reagent. Total RNA was isolated according to the manufacturer's instructions. SYBR-Green PCR Master Mix (Arraystar) was used for qPCR analysis, and all reactions were run on a QuantStudio TM 7 Flex Real-time PCR System (Thermo Fisher Scientific, Inc.) using the following steps: Denaturation step (95°C for 10 min), and 45 cycles of three step amplification (denaturation, 95°C for 10 sec; annealing, 58°C for 5 sec; and extension, 72°C for 10 sec). All RNA expression data were normalized to GAPDH. The relative expression of the genes was calculated using the 2−ΔΔCq method (29 (link)). The primers used in the present study are summarized in Table I .
8
Quantification of Immune Biomarkers via qRT-PCR
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Oligonucleotide primer sets and a TaqMan probe were designed using Primer3 software (Howard Hughes Medical Institute, MA, USA) for performing the qRT-PCR TaqMan probe assay targeting multiple immune markers including IFN-γ, TNF-α, IL-2R, CXCL9, IP-10, CXCL11, CCL11, GM-CSF, and TNFR. The oligonucleotide sequences of the PCR primer pairs and TaqMan probes were based on the National Center for Biotechnology Information (NCBI) reference mRNA or cDNA sequences of human target genes. Quantitative PCR was carried out with THUNDERBIRDTM Probe qPCR Master Mix (TOYOBO, Osaka, Japan) using 3 μL of cDNA as the template in a total volume of 20 μL. The thermal cycling conditions were 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. All reactions were performed using an ABI 7500 FAST Real-time PCR system (Applied Biosystems, Waltham, MA, USA) and QuantSTUDIOTM 7 Flex Real-time PCR system (Thermo Fisher Scientific). Relative quantification was performed by the 2-ddCT method; relative expression was calculated as the ratio between the mean threshold cycle (CT) values of the target genes and reference gene (GAPDH) in each stimulated sample in relation to a reference sample (not stimulated) [11 (link)].
9
Isolation and Quantification of RNA from Cells and Bone
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For isolating RNA from cells, an RNA purification kit for cells (EZBioscience, Cat. B0004DP) was utilized according to the manufacturer’s protocol. For isolating RNA from cortical bone, femurs and tibia were cleaned of soft tissue and bone marrow and crushed in a tissue grinder machine (Servicebio). RNA was isolated from the bone powder using the RNA purification kit for tissue (EZbioscience, Cat. EZB-RN001-plus) according to the manufacturer’s protocol. An additional DNase1 digestion step was performed to ensure that the samples were not contaminated with genomic DNA. RNA concentration was assessed with Nanodrop spectrophotometers (Thermo Fisher Scientific, QuantStudio™ 7 Flex Real-Time PCR System, QuantStudio Real-Time PCR 1.3).
For reverse transcription, 1000 ng RNA was reverse transcribed using 4×Reverse Transcription Master Mix (EZbioscience, Cat. EZB-RT2GQ). qPCR was performed using 2×SYBR Green Color qPCR Mix (EZbioscience, Cat. A0001-R1) following the manufacturer’s recommendation. Samples were tested on a Quant StudioTM 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The results were calculated using the ΔΔCT method and are presented as the x-fold increase relative to GAPDH mRNA levels. Primers were synthesized by BioSune company and are listed in (Supplementary Table1b ).
For reverse transcription, 1000 ng RNA was reverse transcribed using 4×Reverse Transcription Master Mix (EZbioscience, Cat. EZB-RT2GQ). qPCR was performed using 2×SYBR Green Color qPCR Mix (EZbioscience, Cat. A0001-R1) following the manufacturer’s recommendation. Samples were tested on a Quant StudioTM 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The results were calculated using the ΔΔCT method and are presented as the x-fold increase relative to GAPDH mRNA levels. Primers were synthesized by BioSune company and are listed in (Supplementary Table
10
Colon Tissue RNA Extraction and qPCR
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Colon tissue was homogenized by tissue homogenizer (LUKYM-I) and total RNA was isolated using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, RC112–01), and the HiScript III 1st Strand cDNA Synthesis Kit with a gDNA wiper (Vazyme, R312–01) was used to reverse-transcribe the RNA into cDNA. The QuantStudioTM 7 Flex Real-Time PCR System (Thermo Fisher Scientific) was used to measure the relative expression level of the gene and normalize it to the expression level of β-actin. Primers used in this study are listed in Supplementary Table 1.
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