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.
Sybr master mix
Manufactured by Qiagen
Sourced in Germany
SYBR master mix is a pre-formulated solution containing all the necessary components for real-time PCR amplification using SYBR Green dye. It is designed to facilitate the setup and performance of real-time PCR assays.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Goscript reverse transcription system, by Promega (4 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Mastercycler realplex, by Eppendorf (2 mentions)
Sybr green master mix, by Qiagen (2 mentions)
Mircury lna universal rt kit, by Qiagen (2 mentions)
Specific primers, by Qiagen (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Roter gene q system, by Qiagen (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Rotor gene q real time pcr cycler, by Qiagen (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Superscript cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Quantitect, by Qiagen (1 mentions)
Steponeplus real time pcr, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
17 protocols using sybr master mix
1
Transcriptomic Analysis of Hippocampus in Epilepsy
2
Quantitative PCR for Chromatin Immunoprecipitation
3
Quantifying Gene Expression in Rice-Magnaporthe Interaction
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Total RNA was extracted using the leaf tissue infected with M. oryzae strains with an RNeasy Plant Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized from RNA (Takara, Shiga, Japan). qRT-PCR analysis was performed using a Roter-Gene Q system (Qiagen, Hilden, Germany) and 2X SYBR Master mix. Relative expression was calculated with qRT-PCR using the threshold cycle value (CT) of each target gene against the CT value of the rice genomic ubiquitin (OsUbi) gene according the 2−ΔΔCT method [52 (link)]. qRT-PCR was conducted with three replications. The primer sequences used for qRT-PCR analysis are listed in Table S2 .
4
Quantitative PCR analysis of gene expression
5
Quantitative Gene Expression Analysis
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Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen, USA). Reverse transcription was performed according to the standard protocol using the GoScript reverse transcription system (Promega, USA). Quantitative PCR was conducted using SYBR master mix (Qiagen, Germany) on a Roche light 480II system. The mRNA expression levels of several markers were detected via the comparative cycle threshold (Ct) method and normalized to the expression levels of GAPDH. The sequences of primers used for PCR are listed in Table 2 .
Sequences of primers used for PCR
| Gene | Primer |
|---|---|
| GAPDH | 5′-TTGATGGCAACAATCTCCAC-3′ |
| 3′-CGTCCCGTAGACAAAATGGT-5′ | |
| α-SMA | 5′-GTCCCAGACATCAGGGAGTAA-3′ |
| 3′-TCGGATACTTCAGCGTCAGGA-5′ | |
| PDGFR-β | 5′-AGGAGTGATACCAGCTTTAGTCC-3′ |
| 3′-CCGAGCAGGTCAGAACAAAGG-5′ | |
| Collagen I | 5′-ATGGATTCCCGTTCGAGTACG-3′ |
| 3′-TCAGCTGGATAGCGACATCG-5′ | |
| Fibronectin | 5′-GCTCAGCAAATCGTGCAGC-3′ |
| 3′-CTAGGTAGGTCCGTTCCCACT-5′ |
6
Quantitative rRNA Depletion Analysis
7
Quantitative RNA Expression Analysis
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Total RNA was extracted from renal tissues and cells using TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA), and one microgram of RNA was reverse transcribed to first-strand cDNA using the GoScript reverse transcription system (Promega, USA). Quantitative PCR was conducted using SYBR master mix (Qiagen, Germany) on a Roche LightCycler 480II. Relative mRNA expression levels were calculated using the 2-ΔΔCt method and were normalized to the corresponding expression levels of GAPDH. The primer sequences used to amplify the human and mouse RNA are shown in Table 1 .
8
Quantitative Real-time PCR for Gene Expression
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Quantitative Real-time PCR was performed as described previously [28] (link). Briefly, total RNA or miRNA from cells was isolated with RNeasy or miRNeasy Mini kit (QIAGEN). cDNA was synthesized using the SuperScript cDNA synthesis kit (Invitrogen) or High Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, NY) and quantitative RT-PCR (qRT-PCR) reactions were performed using SYBR MasterMix (Qiagen) by StepOnePlus Real-Time PCR System (Applied Biosystems, Foster city, CA) in triplicate. 18S or RNU6B was used to normalize the miRNA and mRNA data, respectively. Expression was calculated using the 2−ΔΔCt method. Primers used are described in File S1 .
9
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using RNeasy kit (Qiagen) from the brain and liver at PD 1 and was reverse-transcribed using cDNA kit according to the manufacturer's instruction (Qiagen). Quantitative real time PCR with 10 ng of cDNA were carried out in duplicate in a 7300 real time PCR system (Effendorff, Hauppauge, NY) using the SYBR master mix (Qiagen) and the following cycles: 2 min at 50°C, 10 min at 95°C, and then 40 cycles each at 95°C for 15 s and 60°C for 60 s [19 (link)]. RT2 qPCR analysis was also carried out according to manufacturer's manual using β-actin as a control. All primers used in this study were purchased from Qiagen. For data analysis the Ct method was used; for each gene fold changes were calculated as difference in gene expression of G3 HO and G3 HOT compared to that in WT. ΔCt was calculated by subtraction of Ct of β-actin from Ct of the specific gene. ΔΔCt was calculated by subtraction of ΔCt of WT from ΔCt of G3 HO or H3 HOT. Fold change was determined by 2ΔΔCt. More than 1 indicates gene upregulation and less than 1 indicates gene downregulation.
10
Quantification of Fibrosis Markers via qRT-PCR
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Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen, USA). One microgram RNA was reverse transcribed into first strand cDNA using the GoScript reverse transcription system (Promega, USA) in a 20 μL reaction system. The cycling parameters were used as follow: 40 cycle pf denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s. Triplicated experiments for each sample were processed. Quantitative PCR was conducted using SYBR master mix (Qiagen, Germany) on the Roche light 480II. The mRNA expression levels of several markers, including TGF-β, fibronectin and Collagen I were conducted via the comparative cycle threshold (Ct) method and normalized to the expression levels of GAPDH. Primer sequences were listed in Table 1 .
Primer sequences for qRT-PCR [52 (link)]
| Genes | Sequences |
|---|---|
| GAPDH | Forward primer: 5′-TTGATGGCAACAATCTCCAC-3′ |
| TGF-β | Forward primer: 5′-CTTCAATACGTCAGACATTCGGG-3′ |
| Fibronectin | Forward primer: 5′-GCTCAGCAAATCGTGCAGC-3′ |
| Collagen I | Forward primer: 5′-ATGGATTCCCGTTCGAGTACG-3′ |
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