Total RNA from mouse liver was isolated using TRIzol (QIAGEN, Hilden, Germany). RNA was quantified using the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) [22 (link)]. To process RNA for real-time quantitative polymerase chain reaction (RT-qPCR), we treated samples with DNase (Applied Biosystems, Darmstadt, Germany). Samples were then converted into cDNA using the reverse transcription kit (QIAGEN, Hilden, Germany). To carry out PCR, the ABI Prism 7500HT sequence detection system (Applied Biosystems, Darmstadt, Germany) with SYBR green PCR master mix from QIAGEN (Hilden, Germany) was used. The following genes were investigated: interleukin-1β (IL-1β) (Mm_Il1b_2_SG, cat. no. QT01048355), interleukin-6 (IL-6) (Mm_Il6_1_SG, cat. no. QT00138663), interferon gamma (INF-γ) (Mm_Ifng_1_SG, cat. no. QT01038821), tumor necrosis factor-alpha (TNF-α) (Mm_Tnf_1_SG, cat. no. QT00104006), Bcl2-associated X protein (Bax) (Mm_Bax_1_SG, cat. no. QT00102536), B-cell lymphoma 2 (Bcl2) (Mm_Bcl2_3_SG, cat. no. QT00156282), and caspase-3 (Casp3) (Mm_ Casp3_1_SG, cat. no. QT00260169). The used primers were purchased from QIAGEN. PCR reaction was performed as described by Dkhil et al. [4 (link)]. The 2−ΔΔCT method was used to determine the fold change in mRNA expression [29 (link)].
Abi prism 7500 ht sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7500 HT Sequence Detection System is a real-time PCR instrument designed for high-throughput nucleic acid analysis. It is capable of performing quantitative and qualitative gene expression, SNP genotyping, and other real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Sybr green pcr mix, by Roche (3 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (3 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Hot firepol evagreen qpcr mix plus rox, by Solis BioDyne (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Accupower rt premix, by Bioneer (2 mentions)
Taqman primer probe kit, by Thermo Fisher Scientific (2 mentions)
Iscript advanced cdna kit, by Bio-Rad (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Trizol, by Qiagen (1 mentions)
Sybr green pcr master mix, by Qiagen (1 mentions)
Reverse transcription kit, by Qiagen (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
18s rrna vic, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
33 protocols using abi prism 7500 ht sequence detection system
1
Quantification of Liver Gene Expression
2
Quantitative Gene Expression Analysis
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Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized using Reverse Transcription Kit (Takara, Dalian, China). Real-time PCR was performed using SYBR Green PCR mix (Roche, Indianapolis, USA) on an ABI Prism® 7500HT Sequence Detection System (Applied Biosystems, CA, USA). The mRNA expression levels of genes of interest were presented as fold exchange that was normalized to β-actin expression in control group. Primer sequences used in the present study are listed in Additional file 2 : Table S1.
3
Quantitative RT-PCR Analysis of Innate Immune Receptors
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The ABI PRISM 7500HT Sequence Detection System (Applied Biosystems, NY) was used for qt-RT-PCR analysis. Primer-probes sets for TLR1, TLR2, TLR4 and PU.1 (all FAMTM) and 18S rRNA VIC® were obtained predesigned from Applied Biosystems and tested for primer efficacy (gene expression assays: Hs99999901_s1 18S VIC®, Hs00413978_m1 TLR1, Hs01872448_s1 TLR2, Hs00152939_m1 TLR4, Hs00162150_m1 PU.1). Multiplex amplification was carried out in a total volume of 20 μl for 40 cycles of 3 seconds at 95°C, 30 seconds at 60°C. Initial denaturation was performed for 3 min at 95°C. Target gene expression was normalized to 18s rRNA house keeping gene expression. Normalized target gene expression was analysed by the comparative ΔΔ-CT method and calculated as x-fold expression.
4
Quantifying VentX mRNA in Monocyte-Derived Macrophages
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For VentX mRNA quantification, 2 × 105 monocytes were left untreated or were differentiated with M‐CSF in the absence or presence of hLF. Cells were harvested on day 6 and total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Venlo, the Netherlands). RNA quantity and quality were assessed using NanoDrop8000 (Thermo Fisher Scientific, Waltham, MA, USA). The ABI PRISM 7500HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used for RT‐qPCR analysis. Primer‐probes sets for VentX FAM and 18S rRNA VIC were obtained predesigned from Applied Biosystems and tested for primer efficacy (gene expression assays: Hs99999901_s1 18S VIC, Hs00797729_s1 VentX FAM). Multiplex amplification was carried out in a total volume of 20 µl for 40 cycles of 3 s at 95°C, 30 s at 60°C. Initial denaturation was performed for 3 min at 95°C. Target gene expression was normalized to 18s rRNA housekeeping gene expression. Normalized target gene expression was analysed by the comparative ΔΔCT method and calculated as x‐fold expression.
5
miRNA Expression Analysis by qRT-PCR
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Total RNA was extracted using the TRIzol reagent (Invitrogen Life Technology, Carlsbad, CA, USA). Single-strand cDNA was synthesized from 2 mg of total RNA with the PrimeScript RT reagent kit (TaKaRa Bio, Dalian, China). For the qRT-PCR analysis, the miRNAs were reverse-transcribed with RT primers (RiboBio, Guangzhou, China). β-actin and U6 were used as loading controls for quantitation of the mRNAs and miRNAs respectively. The Bulge-Loop miRNA qRT-PCR Primer Set (RiboBio) was used for qRT-PCR of miR-705 (product ID: miRQ0003495-1-2) and U6 (Product ID: MQP-0202). All real-time PCR analyses were performed and analysed with the SYBR Premix Ex Taq II kit (TaKaRa) and the ABI Prism 7500 HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The primer sequences are shown in Table S1 .
6
Quantitative Gene Expression Analysis
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Total RNA was extracted with Trizol reagent (Invitrogen, USA), and reverse-transcribed with oligo-dT using the M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s protocol. For semi-quantitative polymerase chain reaction (PCR), the resultant cDNA was subjected to 25–30 cycles of PCR amplification (denaturing at 95 °C for 30 s, annealing at 55–60 °C for 30 s, extension at 72 °C for 60 s). The PCR products were separated by electrophoresis on a 2% agarose gel and visualized with ethidium bromide staining. Quantitative real-time PCR was performed using SYBR green reaction mixture in the ABI 7500 fast real-time PCR system (Applied Biosystems, USA). The PCR conditions were one cycle at 55 °C for 2 min and at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s and at 60 °C for 1 min. The fluorescent signals were detected using the ABI Prism 7500HT sequence detection system (Applied Biosystems, USA). The gene expression data were normalized to the endogenous control β-actin. The relative expression levels of genes were measured according to the formula 2−ΔΔCt, where ΔCt is the difference in threshold cycle values between the targets and β-actin, and ΔΔCt is the difference between the treatment and vehicle control groups. All samples were analyzed in triplicate.
7
Quantitative Analysis of TROP2 Expression
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RNA was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the PrimeScript™ RT Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. The reverse transcription temperature protocol used was 42°C for 50 min and 95°C for 5 min. The primers for TROP2 (All-in-One qPCR Primer) were purchased from GeneCopoeia, Inc., and GAPDH primers (internal control) were purchased from Genscript. The primer sequences were as follows: TROP2 forward, 5′-TGT CCT GAT GTG ATA TGT CTG AG-3′ and reverse, 5′-GGG TGA GAG TGG GTT GGG-3′; and GAPDH forward, 5′-GGA GCG AGA TCC CTC CAA AAT-3′ and reverse, 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′. qPCR was performed on an ABI PRISM 7500HT Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in 96-well plates. The qPCR thermocycling conditions were: Pre-denaturation at 95°C for 10 min, followed by 30 cycles of 95°C for 10 min, 56°C for 30 sec and 72°C for 30 sec; with a final extension step at 72°C for 30 sec. The products were stored at 4°C until further use. Relative expression levels were calculated as ratios normalized to GAPDH. The Cq-value for each sample was calculated using the ΔΔCq method, and the results were expressed as 2-ΔΔCq (21 (link)). Experiments were performed in triplicate.
8
Quantification of mRNA Expression
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cDNAs from total RNA (50 ng for beclin-1, LC3, p62, hsc70 and BDNF and 100 ng for asyn, TDP-43, lamp2A, hsp70) were amplified in triplicate in the ABI Prism 7500 HTSequence Detection System (Applied Biosystems). 5 × HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne) was used at the following conditions: 95 °C for 15 min, 40 cycles of: 95 °C for 15 s, 62.5 °C for 20 s, 72 °C for20s. The sequences of the primers used (Sigma-Aldrich) are listed in Supplementary Table 1 A. To analyze MEF2D mRNA levels, TaqMan Gene Expression Assay (Applied Biosystems, assay ID: Hs00945735_m1; β-actin assay ID: Hs99999903_m1) was used to amplify cDNA (70 ng). For relative quantification of each target vs. β-actin mRNA, the comparative CT method was used.
9
Quantifying Epithelial-Mesenchymal Transition Markers
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Cell RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using a PrimeScript™ RT reagent kit (Takara, Glen Burnie, MD) in accordance with the manufacturer's instructions. The primers of Trop2 were β‐catenin, E‐cadherin, fibronectin, vimentin，goocecoid, and snail. GAPDH (internal control) was purchased from GENEray (shanghai, China). qRT‐PCR was performed on an ABIPRISM 7500HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in 96‐well plates. Relative expression levels were calculated as ratios normalized against those of GAPDH. Results were normalized to respective internal controls. The Ct‐value for each sample was calculated using the ΔΔCt method, and results were expressed as 2−ΔΔCt. Primers can be found in Table S2 .
10
Quantitative RT-qPCR Analysis of IFNA
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The ABI PRISM 7500HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and One‐Step RT‐qPCR kit (Bio‐Rad, Hercules, CA, USA) was used for quantitative RT‐PCR analysis. Primer‐probes sets for IFNA FAM and GAPDH VIC were obtained predesigned from Applied Biosystems and tested for primer efficacy (gene expression assays Hs00265051_s1). Multiplex amplification was carried out in a total volume of 20 µl for 40 cycles of 3 s at 95°C and 30 s at 60°C. Initial denaturation was performed for 3 min at 95°C. Target gene expression was normalized to GAPDH housekeeping gene expression. Normalized target gene expression was analyzed by the comparative ΔΔCT method and calculated as x‐fold expression.
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