Total RNA was prepared from kidneys or cells, using QIAzol® Lysis Reagent (Qiagen). Complementary DNA was synthesized from 1 µg total RNA using oligo(dT) primers (Thermo Fisher Scientific) and ReverTra Ace (Toyobo, Osaka, Japan). Gene expression was quantified using the Fast SYBR™ Green Kit (Applied Biosystems/Thermo Fisher Scientific). The primers used in this study were synthesized by Invitrogen/Thermo Fisher Scientific and are listed in Supplementary Table 1 .
Sybr green fast kit
Manufactured by Thermo Fisher Scientific
The SYBR green fast kit is a reagent used in real-time PCR (polymerase chain reaction) experiments. It contains SYBR green, a fluorescent dye that binds to double-stranded DNA, allowing for the quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Superscript 3 reverse, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Cfx96 touch system, by Bio-Rad (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Transzol up, by Transgene (1 mentions)
Trizol rna extraction method, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
9 protocols using sybr green fast kit
1
Quantitative Gene Expression Analysis
2
Optimized RT-qPCR for Relative Gene Expression
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RNA samples extracted as described above were used with Superscript III reverse transcriptase (Invitrogen) to generate cDNA according to the manufacturer’s instructions. Serial dilutions of 1/8 to 1/256 were used as templates for QRT-PCR. PCR products were gel purified on a 2% Agarose gel and extracted with a Sigma gel extraction kit according to the manufacturer’s instructions and 10-fold serial dilutions of this to from 10−4 to 10−9 were used as template for standard curves. Reactions were carried out with the Fast SYBR green kit (Applied Biosystems) on an Applied Biosystems Quantstudio 6 Flex instrument run by Quantstudio Real Time PCR software v 1.3. Cycling conditions were 1 cycle at 95°C for 20 s followed by 40 cycles of 95°C for 1 s and 60°C for 20 s, then a melt curve of 95°C for 20 s, then 60°C for 60 s and 95°C for 15 s. Primer concentrations were optimized for each template at 30–200 nM and reaction efficiencies were monitored in the software. Only reactions with 90–110% efficiency were used and expression levels were output relative to the standard curve and normalized relative to all reactions per biological repeat.
3
Macrophage Differentiation and Gene Expression
4
Nanoparticle-Induced Macrophage Gene Expression
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Monocytes were isolated from the whole blood of healthy volunteers (UCSF Institutional Review Board-approved protocol 16-20174) using density gradient centrifugation and differentiated into macrophages in Roswell Park Memorial Institute (RPMI) medium (ThermoFisher) supplemented with 5% FBS and granulocyte-macrophage colony-stimulating factor (10 ng/mL) for 7 days. After differentiation, the medium was replaced and macrophages were treated with vehicle or 1010 nanoparticles per milliliter for 24 hours, at which point the cells were lysed with buffer RLT (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol. Total RNA was isolated using an RNeasy Micro Kit (Qiagen) and quantified using a NanoDrop 2000 (ThermoFisher). Complementary DNA (cDNA) was made by reverse transcription-polymerase chain reaction (PCR) using 1 μg of RNA. Real-time PCR was performed using a Fast SYBR Green kit (Life Technologies, Carlsbad, Calif) to assay for expression of tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), C-X-C motif chemokine 10 (CXCL10), interleukin 10 (IL-10), chemokine (C-C motif) ligand 17 (CCL17), and mannose receptor C-type 1 (MRC1) genes (see Supplementary Table , for primer sequences). Data were normalized to two housekeeping genes, hypoxanthine-guanine phosphoribosyltransferase and ubiquitin C, and then to healthy individuals using the 2−ΔΔCt method.
5
Quantitative Analysis of Upd Expression
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RNA was isolated using TransZol Up (Transgen, Beijing, China). cDNA was synthesized from 1 μg of RNA with the PrimeScriptTM RT-PCR Kit (Takara, Kyoto, Japan). Analysis was performed in a CFX96 Touch system (Bio-Rad, Hercules, California) using SYBR Green Fast kit (Applied Biosystems, Waltham, Massachusetts). rp49 expression was used for normalization. Three experiments per genotype were averaged. A biological replicate was performed with same results. Primers used were:
upd-For: 5′ TCCACACGCACAACTACAAGTTC 3′;
upd-Rev: 5′ CCAGCGCTTTAGGGCAATC 3′;
upd2-For: 5′ AGTGCGGTGAAGCTAAAGACTTG 3′;
upd2-Rev: 5′ GCCCGTCCCAGATATGAGAA 3′;
upd3-For: 5′ TGCCCCGTCTGAATCTCACT 3′;
upd3-Rev: 5′ GTGAAGGCGCCCACGTAA 3′;
rp49-For: 5′ GGCCCAAGATCGTGAAGAAG 3′;
rp49-Rev: 5′ ATTTGTGCGACAGCTTAGCATATC 3′.
upd-For: 5′ TCCACACGCACAACTACAAGTTC 3′;
upd-Rev: 5′ CCAGCGCTTTAGGGCAATC 3′;
upd2-For: 5′ AGTGCGGTGAAGCTAAAGACTTG 3′;
upd2-Rev: 5′ GCCCGTCCCAGATATGAGAA 3′;
upd3-For: 5′ TGCCCCGTCTGAATCTCACT 3′;
upd3-Rev: 5′ GTGAAGGCGCCCACGTAA 3′;
rp49-For: 5′ GGCCCAAGATCGTGAAGAAG 3′;
rp49-Rev: 5′ ATTTGTGCGACAGCTTAGCATATC 3′.
6
Quantifying Gene Expression in Drosophila Clones
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Total RNA from Eye-antenna imaginal discs containing wild-type or mutant clones was isolated using a Trizol RNA extraction method (Invitrogen). The SuperScriptIII First-Strand Synthesis System (Invitrogen) kit was used to synthesize complementary DNA. Real-time PCR was carried out on an Applied Biosystems machine using the SYBR green fast kit (Applied Biosystems) following the manufacturer's instructions. Relative gene expression was obtained from triplicate runs normalized to Rp49 as endogenous control. The following primers were used: Rp49, 5′-GGCCCAAGATCGTGAAGAAG-3′ and 5′-ATTTGTGCGACAGCTTAGCATATC-3′; Spitz, 5′-CGCCCAAGAATGAAAGAGAG-3′ and 5′-AGGTATGCTGCTGGTGGAAC-3′
Clones of mutant cells in the eye-antennal discs were generated using standard MARCM system56 (link). Immunostaining and Immunoprecipitation experiments were performed as previously described57 (link).
Clones of mutant cells in the eye-antennal discs were generated using standard MARCM system56 (link). Immunostaining and Immunoprecipitation experiments were performed as previously described57 (link).
7
Quantifying Transcriptional Changes in Drosophila Tumor Models
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Total RNA from wild-type and tumor imaginal discs (n ≥ 30 pairs) were extracted using Trizol. cDNAs were synthesized from 500 ng total RNA with the iScript cDNA Synthesis Kit (Bio-Rad). cDNAs were subjected to real-time PCR with the SYBR Green Fast Kit (Applied Biosystems), according to the manufacturer’s instructions. The expression level of genes for each sample was calculated by comparing to the internal control, rp49. The relative fold change of each gene was normalized to the expression level of the same gene in the eye disc bearing RasV12. Three experiments for each condition were averaged. The following primers were used for qRT-PCR:
upd: 5′-TCCACACGCACAACTACAAGTTC-3′; 5′-CCAGCGCTTTAGGGCAATC-3′
upd2: 5′-AGTGCGGTGAAGCTAAAGACTTG-3′; 5′-GCCCGTCCCAGATATGAGAA-3′
upd3: 5′-TGCCCCGTCTGAATCTCACT-3′; 5′-GTGAAGGCGCCCACGTAA-3′
dp53: 5′-CTATTGAGCTGGCGTTCGTCTTGGAT-3′; 5′-TCTGCCAAAACTCGTGTATCGGGCG-3′
dap/p21: 5′-GTCAGCTTCCAGGAGTCGAG-3′; 5′-CCAAAGTTCTCCCGTT CTGA-3′
rp49: 5′-GGCCCAAGATCGTGAAGAAG-3′; 5′-ATTTGTGCGACAGCTTAGCATATC-3′
upd: 5′-TCCACACGCACAACTACAAGTTC-3′; 5′-CCAGCGCTTTAGGGCAATC-3′
upd2: 5′-AGTGCGGTGAAGCTAAAGACTTG-3′; 5′-GCCCGTCCCAGATATGAGAA-3′
upd3: 5′-TGCCCCGTCTGAATCTCACT-3′; 5′-GTGAAGGCGCCCACGTAA-3′
dp53: 5′-CTATTGAGCTGGCGTTCGTCTTGGAT-3′; 5′-TCTGCCAAAACTCGTGTATCGGGCG-3′
dap/p21: 5′-GTCAGCTTCCAGGAGTCGAG-3′; 5′-CCAAAGTTCTCCCGTT CTGA-3′
rp49: 5′-GGCCCAAGATCGTGAAGAAG-3′; 5′-ATTTGTGCGACAGCTTAGCATATC-3′
8
cDNA Synthesis and Real-Time PCR
9
Quantifying dTRAF1 Expression in Drosophila
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Total RNA was extracted from third instar wing discs from ptc-Gal4 or ptc-Gal4, UAS-CiRNAi animals using a standard TriZol extraction. RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) according to manufacturer’s instructions. dTRAF1 expression was quantified relative to Rp49 (RpL32- FlyBase, endogenous control) by real-time PCR performed in triplicate using the SYBR Green fast kit (Applied Biosystems) and an Applied Biosystems machine according to the manufacturer’s instructions. The following primers were used: dTRAF1, 5’-GCACTCCATCACCTTCACAC-3’ and 5’-TAGCTGATCTGGTTCGTTGG-3’; Rp49, 5′-GGCCCAAGATCGTGAAGAAG-3′ and 5′-ATTTGTGCGACAGCTTAGCATATC-3′.
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