RNA from mouse liver and small intestine was isolated by RNeasy Mini Kit, RNA from mouse brown adipose tissue was isolated by RNeasy Lipid Tissue Mini Kit (QIAGEN GmbH, Hilden, Germany). Subsequent cDNA synthesis was carried out using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Vilnius, Lithuania), and real-time PCR using specific primers (QIAGEN GmbH, Hilden, Germany) for mouse Ces1, Ces2, Ces3 genes, thermogenic genes, hepatic fatty acid and triglyceride synthesis genes were performed as described previously25 (link),26 (link).
Specific primers
Manufactured by Qiagen
Sourced in Germany
Specific primers are short DNA sequences designed to precisely target and amplify specific regions within a larger DNA molecule. They serve as essential components in various molecular biology techniques, such as polymerase chain reaction (PCR), by providing the necessary starting points for DNA synthesis and replication.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Omniscript rt pcr kit, by Qiagen (2 mentions)
Abi 7500 system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix kit, by Takara Bio (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Sybr master mix, by Qiagen (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Facsaria 5, by BD (1 mentions)
Rt2 sybr green fast mastermix, by Qiagen (1 mentions)
Macs mrna isolation kit, by Miltenyi Biotec (1 mentions)
Lysis binding buffer, by Miltenyi Biotec (1 mentions)
Iq5 real time pcr detection system, by Bio-Rad (1 mentions)
Sensifast sybr, by Meridian Bioscience (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Dnase 1, by Qiagen (1 mentions)
15 protocols using specific primers
1
Transcriptional Profiling of Metabolic Genes
2
Transcriptomic Analysis of Hippocampus in Epilepsy
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Hippocampus samples from VEH and RESV treated groups collected 1 and 4 days after SE were also used for measuring the expression of select genes related to inflammation, longevity and cognition, along with samples from age-matched naive control animals (n = 4-6/group). The total RNA and cDNA were prepared as described above. The template cDNA was next amplified separately using specific primers (Qiagen, Valencia, CA) of multiple genes. This comprised genes linked to inflammation (interleukin-1beta [IL-1β], TNFα, nuclear factor kappa B [NFκB], interferon-gamma [IFN-γ], IL-4 and IL-10, myeloperoxidase [MPO]), and longevity and cognition (NAD-dependent deacetylase sirtuin-1 [SIRT1], Forkhead box O3 [FOXO3]). In brief, for each qPCR reaction, 1 μl template cDNA was mixed with 12.5 μl of 2X SYBR master-mix (SABiosciences, Qiagen, Valencia, CA), 1 μl of gene specific primer (RT2 qPCR primer mix containing 10 μM each of forward and reverse primers, Qiagen, Valencia, CA) and 10.5 μl of dH2O for a total volume of 25 μl. Each assay also comprised two housekeeping genes namely GAPDH and Act B. After a brief centrifugation, reactions were carried out using a CFX96 Real-Time system (Bio-Rad, Hercules, CA). Data collection and analyses were performed as described above.
3
Oligodendrocyte Lineage Progression Analysis
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Brains from 40 adult naïve C57BL/6 mice were extracted, pooled, dissociated using Accutase, and purified using 40% Percoll as described above. Cells were counted, and an aliquot was removed for control single stains and FMO tubes. Fc receptors were blocked, and live cells were labeled with Calcein Violet AM as before. Cells were then stained with a cocktail of anti-A2B5, PDGFRα, NG2, O4, GALC, and MOG in flow cytometry buffer for 30 min at 4°C. Cells were washed twice and resuspended in flow cytometry buffer. Early OPCs (A2B5+PDGFRα+), intermediate OPCs (O4+NG2+), and mature oligodendrocytes (GALC+MOG+) were sorted by FACS into lysis/binding buffer (Miltenyi Biotec) using a FACSAria 5 (BD Biosciences). Sorted cells were stored at −80°C. After thawing on ice, total RNA was isolated using µMACS mRNA Isolation Kit (Miltenyi Biotec) and cDNA was synthesized on column with µMACS One-step cDNA Synthesis Kit (Miltenyi Biotec). Gene expression was analyzed by qRT-PCR using specific primers (Qiagen, Valencia, CA, Table S2
4
Cytokine mRNA Expression in Colonic Tissue
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Total RNA was isolated from colonic tissue with the RNA-Micro-kit [Machery-Nagel] according to the manufacture´s protocol. Total RNA [1 μg] was used to synthesise cDNA using iScript [Bio-Rad]. Quantitative real-time polymerase chain reaction [ RT-PCR] was performed with SensiFAST SYBR [Bioline] and specific primers [Qiagen] on CFX-instruments [Bio-Rad]. Using 18S-rRNA, the relative expression level of cytokine mRNA was calculated with the formula: relative cytokine mRNA expression = 2^(ct[mRNA of interest] – ct[mRNA 18SrRNA]).
5
PD-1/PD-L1/PD-L2 Expression Analysis in GC
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Among the 340 GC patients for whom IHC staining was conducted, total mRNA was extracted from 21 patients’ tumor tissues and paired adjacent non-tumorous tissues (located > 3 cm away from the tumor and confirmed by the pathologists with H&E staining). After reverse transcription, cDNAs were amplified in the presence of primers, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference gene. The expression of PD-1, PD-L1, and PD-L2 was quantified using specific primers (Qiagen), and the mRNA levels of these three genes were analyzed with the 2−ΔΔCt method.
6
Validation of mRNA Microarray Using RT-qPCR
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To validate the mRNA microarray results, three significantly upregulated genes were selected and their expression was analysed with real-time quantitative polymerase chain reaction (RT-qPCR) (Bio-Rad, Stockholm, Sweden). Skin explants from facial or breast skin from nine patients with a median age of 37 years were used for this conformational study. It is noteworthy that these donors differ from the donors used for the micro-array study. Total RNA from these skin explants treated with RA, as described above, were prepared with RNeasy mini kit (Qiagen) and treated with DNase I (Qiagen) to remove contaminating genomic DNA. Reverse transcription was performed with iScript cDNA synthesis kit (Bio-Rad). SsoFast EvaGreen supermix (Bio-Rad) was used in the RT-qPCR together with specific primers (from Qiagen) for the genes of interest, that is, DHRS9, CYP26B1 and HBEGF. Hot-start two-step PCR, consisting of 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 57°C for 30 s was performed using CFX96 Real-Time PCR Detection System (Bio-Rad). Relative quantification of gene expression in the different explants was calculated using the delta-delta-CT method using the TATA box-binding protein (TBP) as a reference gene.
7
Quantitative RT-PCR Analysis of Gene Expression
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The detailed procedure was conducted as our previous description [22 ]. Total RNAs were extracted from mouse kidney samples by the Trizol reagent. Then, we measured RNA concentrations using a spectrophotometer (One Drop, OD-1000+). Next, extracted RNAs were reverse transcribed with the Omniscript RT-PCR kit (Qiagen, Germany), and amplified products by specific primers (Sangon, Shanghai) were quantified following the manufacturer's protocol. Primer sequences are provided in Table 1 . The PCR products were analyzed by electrophoresis using 2% agarose gels, and the density of the bands was used to quantify the mRNA using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.
8
Quantitative miRNA Analysis from Serum and Cells
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miRNAs were isolated from serum by the miRNeasy Serum/Plasma Advanced Kit and from ECs by MasterPure™ Complete DNA & RNA Purification Kit (Lucigen), retrotranscribed using the miRCURY LNA RT Kit, and quantified by qPCR with the miRCURY LNA SYBR Green PCR Kit (all from Qiagen) as their protocols indicate. Specific primers (Qiagen) were used to measure miRNAs of interest, and the 2‐ΔΔCT method was applied to quantitate the different data15 (link) obtained in the LightCycler 96 Real‐Time PCR System (Roche Life Science).
9
RT-qPCR Validation of RNA-seq and miRNA Expression
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RNA-seq results were validated by RT-qPCR on specific target genes. First-strand cDNA was synthesized from 0.5 μg of total RNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s instructions with previously described primer sequences [21 (link),26 (link)]. The QuantStudio 6 Flex Real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) was utilized for cDNA amplification with Power SYBR® Green Master Mix (Invitrogen, Carlsbad, CA, USA) under the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 1 min. Relative mRNA levels were determined using the comparative Ct method, and were normalized against β-actin or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level.
RT-qPCR was used to validate miRNA expression changes. miRNAs were isolated from HDFs using a miRNAeasy mini kit (Qiagen, Hilden, Germany), and cDNA were synthesized using a miScript II RT kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. miRNAs were detected using specific primers (Qiagen, Hilden, Germany). SNORD68, small nuclear RNA, was used as an internal control for normalization. RT-qPCR was performed to detect the levels of specific miRNAs using a miScript SYBR Green PCR kit (Qiagen, Hilden, Germany). Quantification was carried out using a QuantStudio 6 Flex Real-time PCR system.
RT-qPCR was used to validate miRNA expression changes. miRNAs were isolated from HDFs using a miRNAeasy mini kit (Qiagen, Hilden, Germany), and cDNA were synthesized using a miScript II RT kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. miRNAs were detected using specific primers (Qiagen, Hilden, Germany). SNORD68, small nuclear RNA, was used as an internal control for normalization. RT-qPCR was performed to detect the levels of specific miRNAs using a miScript SYBR Green PCR kit (Qiagen, Hilden, Germany). Quantification was carried out using a QuantStudio 6 Flex Real-time PCR system.
10
Quantitative RT-PCR Analysis of Gene Expression
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The detailed procedure was conducted as our previous description [22 ]. Total RNAs were extracted from mouse kidney samples by the Trizol reagent. Then, we measured RNA concentrations using a spectrophotometer (One Drop, OD-1000+). Next, extracted RNAs were reverse transcribed with the Omniscript RT-PCR kit (Qiagen, Germany), and amplified products by specific primers (Sangon, Shanghai) were quantified following the manufacturer's protocol. Primer sequences are provided in Table 1 . The PCR products were analyzed by electrophoresis using 2% agarose gels, and the density of the bands was used to quantify the mRNA using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.
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