RNA was extracted from cells using a High Pure RNA Isolation kit (Roche) according to the manufacturer’s instructions. Reverse transcription was performed with 4 μl of ReverTra Ace (Toyobo, Osaka, Japan) in a total volume of 20 μl. Subsequently, 1 μl of cDNA fragments was amplified using 10 μl of real-time PCR Master Mix (Toyobo) with 1 μl of TaqMan probes (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 20 μl. Fluorescence from the TaqMan probe for each gene was detected using a QuantStudio 12K Flex (Applied Biosystems, Foster City, CA, USA). The mRNA expression level of each gene was normalized to the 18S rRNA expression level (Mm03928990_g1, Thermo Fisher Scientific). The following TaqMan probes (Thermo Fisher Scientific) were used for quantitative PCR: Cd274 (Mm00452054_m1) and Egr1 (Mm00656724_m1).
Taqman probe
Manufactured by Thermo Fisher Scientific
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TaqMan probes are a type of fluorescent DNA probe used in quantitative real-time PCR (qPCR) experiments. They consist of a sequence-specific oligonucleotide labeled with a fluorescent reporter dye and a quencher dye. During the qPCR process, the probe hybridizes to a target DNA sequence, allowing the reporter dye to emit a fluorescent signal that is proportional to the amount of target DNA present in the sample.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (508 mentions)
Rneasy mini kit, by Qiagen (414 mentions)
Trizol reagent, by Thermo Fisher Scientific (358 mentions)
Trizol, by Thermo Fisher Scientific (319 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (241 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (191 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (155 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (146 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (137 mentions)
Rneasy kit, by Qiagen (109 mentions)
Rneasy plus mini kit, by Qiagen (103 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (101 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (100 mentions)
Iscript cdna synthesis kit, by Bio-Rad (98 mentions)
Nanodrop, by Thermo Fisher Scientific (98 mentions)
Quantitect reverse transcription kit, by Qiagen (84 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (82 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (79 mentions)
Superscript 3, by Thermo Fisher Scientific (72 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (70 mentions)
2 822 protocols using taqman probe
1
Quantitative RT-PCR Analysis of Cd274 and Egr1
2
Real-time PCR and ddPCR Analysis
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For Real-time PCR analysis, 1 µg of total RNA was reverse-transcribed into cDNA using SuperScript IV VILO Master Mix (Thermo Fisher Scientific). TaqMan PCR analysis was performed with TaqMan Gene Expression Master Mix by means of ABI PRISM 7900HT System (Applied Biosystems, Monza, Italy). Taqman probes (Thermo Fisher Scientific) were as following: CDKN2A (Hs00923894_m1), APEX2 (Hs00205565_M1). Fluorescence data were automatically converted into Ct (cycle threshold) values. Data export (threshold 0.20) and analysis was performed by Microsoft Office Excel. Expression data were normalized to the geometric mean of housekeeping genes. For housekeeping genes, the Taqman probes (Thermo Fisher Scientific) were as following: B2M (Hs00984230_m1), UBC (Hs00824723_m1), GAPDH (Hs99999905_m1), ACTB (Hs99999903_m1).
The search for CDKN2A gene copy number was carried out by droplet digital PCR (ddPCR) as follows: DNA isolated from FFPE tumor tissues (as described above) was amplified using ddPCR Supermix for Probes (Bio-Rad, Hercules, CA, USA), using CDKN2A and housekeeping genes (EIF2C1, AP3B1, RPP30) probes (Bio-Rad, Segrate, Italy), according to manufacturer’s protocols as described in (35 (link)).
The search for CDKN2A gene copy number was carried out by droplet digital PCR (ddPCR) as follows: DNA isolated from FFPE tumor tissues (as described above) was amplified using ddPCR Supermix for Probes (Bio-Rad, Hercules, CA, USA), using CDKN2A and housekeeping genes (EIF2C1, AP3B1, RPP30) probes (Bio-Rad, Segrate, Italy), according to manufacturer’s protocols as described in (35 (link)).
3
Quantifying mRNA and microRNA Expression
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The expression of mRNA and microRNA were examined by quantitative PCR analysis using a Quantstudio PCR machine (Thermo Fisher Scientific) and TaqMan probes (Supplemental Table S2 ). Fgf21 mRNA expression was measured using Thermo Fisher TaqMan probes (Supplemental Table S2 ) in studies of Fgf21+/+ cells and mice. Studies of Fgf21 mRNA expression in mice with floxed Fgf21 alleles were performed using Universal ProbeLibrary Probe 67 (Roche, Cat# 4688651001) and amplimers 5′-AGATGGAGCTCTCTATGGATCG-3′ and 5′-GGGCTTCAGACTGGTACACAT-3′ (Eurofins Genomics LLC). Normalized relative mRNA and microRNA expression was performed by measurement of the amount of 18S RNA and U6 RNA, respectively, in each sample.
4
Mouse Brain Tissue RNA Extraction and qRT-PCR
5
Relative quantitation of key genes
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Single-stranded cDNA was synthesized using the TranScriba Kit (A&A Biotechnology, Gdansk, Poland). Relative quantitation was performed for WDR13, THOC2, NCBP1, FMR1, CAMK2A, and SYN1 genes. The duplex qRT-PCR reaction was conducted with an ABI7300 Genetic Analyzer (Applied Biosystems) using the FAM-labeled target TaqMan Probes (Thermo Fisher Scientific, Waltham, MA, USA, Table 1 ) and VIC-labeled reference TaqMan Probes (Table 1 ). Gene expression values were normalized to the expression of the reference genes. Normalized expression of genes in unaffected control was set to 1 and the ΔCt was calculated to determine the fold change of genes in affected patients. Expression data reflect the means of three independent experiments, each performed in triplicate.
6
Quantification of GPR110 and TNF mRNA
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Mice were anesthetized with isoflurane and transcardially perfused with 0.1 M phosphate buffer (pH 7.4). The cerebral cortex was rapidly dissected, and total RNA was extracted by Trizol reagent (Thermo Fisher Scientific, cat# 15596026, MA, USA), reverse transcribed to cDNA with High Capacity cDNA Reverse Transcription kit (Applied Biosystem, cat# 4368814, CA, USA). Quantitative RT-PCR was performed using TaqMan™ probes (Thermo Fisher Scientific) run on the QuantStudio 3 Real-Time PCR system (Applied Biosystems by Thermo Fisher Scientific). cDNA samples were reacted with the TaqMan™ probe for GPR110 (Assay ID: Mm00505409_m1, cat# 4331182) or TNF (Assay ID: Mm00443258_m1, cat# 4331182) using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, cat# 4444557) in the presence of the TaqMan™ probe for HPRT (hypoxanthine guanine phosphoribosyl transferase) (Assay ID: Mm00446968_m1, cat# 4448490) which was used as an internal control. Data were analyzed using comparative Ct method. The relative mRNA level of target gene normalization to HPRT was calculated as 2-ΔΔCt value.
7
Quantification of Fibrotic Gene Expression
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In the mouse model, RNA was isolated (Zymo Research Kit, R1065) from a snap-frozen and homogenized lobe from the right lung. RNA was reverse transcribed to cDNA using iScript Reverse Transcription kit (Bio-Rad), and Taqman probes (Thermo Fisher Scientific) were used to measure the quantity of transcript for Cdkn1a/p21 (Mm04205640_g1), Col3a1 (Mm00802300_m1), Col1a1 (Mm00801666_g1), Col1a2 (Mm00483888_m1), and Ccl2 (Mm00441242_m1).
For human cells, RNA was isolated as described below and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Taqman probes were used to measure the quantity of transcript for COL3A1 (Hs00943809_m1), COL1A1 (Hs00164004_m1), CDKN1A/p21 (Hs00355782_m1), GDF15 (Hs00171132_m1), and TGFB1 (Hs00998133_m1).
Transcript levels of genes of interest were measured by quantitative PCR (qPCR) using QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific) and were normalized to housekeeping gene mRNA, 18S (Hs99999901_s1) in murine samples and GAPDH (Hs03929097_g1) or HPRT (hs02800695_m1) in human samples.
For human cells, RNA was isolated as described below and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Taqman probes were used to measure the quantity of transcript for COL3A1 (Hs00943809_m1), COL1A1 (Hs00164004_m1), CDKN1A/p21 (Hs00355782_m1), GDF15 (Hs00171132_m1), and TGFB1 (Hs00998133_m1).
Transcript levels of genes of interest were measured by quantitative PCR (qPCR) using QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific) and were normalized to housekeeping gene mRNA, 18S (Hs99999901_s1) in murine samples and GAPDH (Hs03929097_g1) or HPRT (hs02800695_m1) in human samples.
8
Quantitative Gene Expression Analysis in Mouse Retina
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Following purification of RNA, cDNA was synthesized using Tetro cDNA Synthesis Kit (Bioline, London, UK) according to the manufacturer's protocol. A 20 ml reaction mixture was used in conjunction with 1 mg RNA, 500 ng Oligo dT primer and 200U reverse transcriptase. Gene expression was measured using mouse specific TaqMan probes (ThermoScientific, MA, USA), as shown in Table 2. The TaqMan probes, cDNA and TaqMan Gene Expression Mastermix (ThermoScientific), were plated in a 384-well transparent plate. The amplification of each sample was performed in technical duplicates, carried out using a QuantStudio 12 K Flex RT-PCR machine (ThermoScientific). Relative fold change was expressed as a percentage change compared to the dim-reared control and normalised to a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Significance for each gene was determined using one-way ANOVA with Tukey's multiple comparison post-test (n ¼ 6 per experimental group). For each analysis, differences with a p < 0.05 were considered statistically significant.
9
Gene Expression Analysis by RT-qPCR
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RNA was isolated using GenElute Mammalian Total RNA Purification Kit (cat. no. RTN350; Sigma-Aldrich) according to the manufacturer’s instructions. cDNA synthesis from isolated RNA was done using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. Synthesized cDNA was used to perform RT-qPCR by using TaqMan Fast Advanced Mastermix (cat. no. 4444557; Life Technologies) and TaqMan probes (Applied Biosystems) or SYBR Green-based qPCR was performed with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on an Applied Biosystems StepOnePlus cycler. TaqMan probes used were SCD4: Hs01120908_m1; ANGPTL4: Hs01101123_g1; AXIN2: Hs00610344_m1; RNS18S: Hs03928985_g1; Axin2: Mm00443610_m1; Sdc4: Mm00488527_m1; and Actb: Mm00607939_s1. All the TaqMan probes were purchased from Thermo Fisher Scientific. Gene expression levels were analyzed based on the ΔΔCt relative quantification method.
10
Retinal Gene Expression Profiling by qRT-PCR
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Total RNA was extracted from the retina using the miRNeasy Plus Mini Kit (QIAGEN) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed in a 10 μL reaction mixture using SuperScript III (Thermo Fisher Scientific), and qRT-PCR was performed (7500 Fast Real-Time PCR System; Thermo Fisher Scientific). The 20 μL qRT-PCR mixture contained 1.0 μL of cDNA, 1.0 μL of TaqMan probe (Thermo Fisher Scientific), and 1× TaqMan Fast Universal PCR Master mix (Thermo Fisher Scientific). PCR was performed in 96-well plates using an initial denaturation step of 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 20 s. For relative comparison of gene expression, we analyzed qRT-PCR data using the comparative Ct method (2-ΔΔCT) normalized to Gapdh as the endogenous control. The following TaqMan probes were used: Atf4 (Mm00515324; Thermo Fisher Scientific), BDNF (Hs02718934; Thermo Fisher Scientific), Ho-1 (Mm00516005; Thermo Fisher Scientific), Nfkb1 (Mm00476379; Thermo Fisher Scientific), NRF2 (Hs00975961; Thermo Fisher Scientific), Gapdh (Mm99999915; Thermo Fisher Scientific), Brn3a (Mm02343791; Thermo Fisher Scientific), Brn3b (Mm00454754; Thermo Fisher Scientific), Thy1 (Mm00493681; Thermo Fisher Scientific), Tgfb1 (Mm00441729; Thermo Fisher Scientific), and p53 (Mm01731290; Thermo Fisher Scientific).
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