Total RNA from cells was obtained using Trizol (Life Technologies) and the total RNA from extracellular vesicles was isolated using the Total EVs RNA & Protein Isolation Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. RNA preparations were treated with “Ready-to-go you-prime first-strand beads” (GE Healthcare) to obtain cDNA. Quantitative real-time PCR was performed using DNA Master SYBR Green I mix (Applied Biosystems). mRNA expression levels in cells were quantified applying the ΔCt method, ΔCt = (Ct of the gene of interest - Ct of Actin). mRNA loading in EVs were quantified applying the ΔCt method but EZH2 and GLI1 levels were normalized using U6Sn levels as internal control. EZH2 and GLI1 primers were obtained from the PrimerBank database (http://pga.mgh.harvard.edu/primerbank/ ).
Dna master sybr green 1 mix
Manufactured by Thermo Fisher Scientific
The DNA Master SYBR Green I mix is a ready-to-use solution for real-time PCR amplification and quantification of DNA. It contains SYBR Green I dye, which binds to double-stranded DNA, and all necessary components for PCR.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (10 mentions)
Ready to go you prime first strand beads, by GE Healthcare (5 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Abi prism 7700, by Thermo Fisher Scientific (2 mentions)
Total evs rna protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Ready to go you prime first strand beads kit, by GE Healthcare (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Rna cleanup, by Qiagen (1 mentions)
Dnase 1 treatment, by Qiagen (1 mentions)
10 protocols using dna master sybr green 1 mix
1
Quantifying mRNA in Cells and Extracellular Vesicles
2
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was obtained using Trizol (Life Technologies) following the manufacturer's instructions. RNA preparations (2 μg) were treated with “Ready-to-go you-prime first-strand beads” (GE Healthcare) to generate cDNA. qRT-PCR was carried out using DNA Master SYBR Green I mix (Applied Biosystems). mRNA expression levels were quantified applying the ΔCt method, ΔCt = (Ct of gene of interest - Ct of GAPDH). qRT-PCR primer sequences were obtained from the PrimerBank data base (http://pga.mgh.harvard.edu/primerbank/ ) (See Table 1 ):
3
Quantitative Real-Time RT-PCR Protocol
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For real-time quantitative RT–PCR, total RNA from cells was extracted with Trizol (Life Technologies). Reverse transcription was performed using random priming and Superscript Reverse Transcriptase (Life Technologies) according to the manufacturer's guidelines. Real-time PCR was performed using an ABI PRISM 7700 (Applied Biosystems) by using DNA Master SYBR Green I mix (Applied Bio-systems). The primer sequences used were previously described [45 (link)].
4
Quantitative RT-PCR for mTRF1, GAPDH, and TopoIIα
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Total RNA from cells was extracted with Trizol (Life Technologies). Samples were treated with DNase I before reverse transcription, using random priming and iScript™ (BioRad) according to the manufacturer’s protocols or using Ready-To-Go You-Prime First-Strand Beads Kit (GE Healthcare). Quantitative real-time PCR was performed using an ABI PRISM 7700 (Applied Biosystems), using DNA Master SYBR-Green I mix (Applied Biosystems) according to the manufacturers protocol. All values were obtained in triplicates. Primers used are as follows: mTRF1-F: 5′-GTCTCTGTGC CGAGCCTTC-3′; mTRF1-R: 5′-TCAATTGGTA AGCTGTAAGT CTGTG-3′; Gapdh-F, 5′-TTCACCACCA TGGAGAAGGC-3′; Gapdh-R, 5′-CCCTTTTGGC TCCACCCT-3′, TopoIIα (a)_Forward 5′-TGGTCAGTTT GGAACCAGGC-3′ TopoIIα (a)_Reverse 5′-TCAGGCTCAA CACGTTGGTT-3′ TopoIIα (b)_Forward 5′-AACGAGAGAC ACATCATTGT CAG-3′ TopoIIα (a)_Reverse 5′-TCACCTTCCC TATCACAGTC C-3′
5
Quantitative Real-Time PCR Methodology
6
Quantitative Real-Time PCR for Gene Expression
7
Quantitative Real-Time PCR for Gene Expression
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Quantitative real time-PCR (qRT-PCR) was carried out using DNA Master SYBR Green I mix (Applied Biosystems), as we previously described (Villasante et al., 2014 (link)) mRNA expression levels were quantified applying the ΔCt method, ΔCt = (Ct of gene of interest—Ct of GAPDH). qRT-PCR primer sequences that were obtained from the PrimerBank data base1 are listed in Table 1 . Primer sequences for CD206, CD163, CCR7, CD80, and TNFα were obtained from Spiller et al. (2014) .
8
Quantitative Real-Time PCR for G6PD Expression
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Total RNA from tissues or cells was extracted using TRIZOL (Life Technologies) followed by RNA cleanup (Qiagen, 74104) and DNase I treatment (Qiagen) in column. Samples were reverse transcribed using random priming and Superscript Reverse Transcriptase (Life Technologies), according to the manufacturer's instructions. Quantitative real-time PCR was performed using DNA master SYBR Green I mix (Applied Biosystems) in an ABI PRISM 7700 thermocycler. Quantifications were made applying the ΔCt method (ΔCt=(Ct of gene of interest−Ct of housekeeping)). The G6PD fold expression corresponds to the sum of the 2(−ΔCt) for the mouse G6PD and human G6PD primers. The housekeeping gene used for input normalization was β-actin. Primer sequences are described in Supplementary Table 1 .
9
Quantitative Analysis of RNA and mtDNA
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For RNA analysis, total RNA from cells was extracted using TRIzol (Life Technologies) and samples were reverse transcribed using random priming and Superscript Reverse Transcriptase (Life Technologies), according to the manufacturer's instructions. qPCR was performed using DNA master SYBR Green I mix (Applied Biosystems) in an ABI PRISM 7700 thermocycler and applying the 2(−ΔΔCt) method. mitochondrial DNA (mtDNA) determination was performed as in refs. 3, 25 (link) using phenol:chloroform:isoamyl alcohol (Sigma) extraction and human mitochondrial ND1 (mtND1) relative to nuclear β2-microglobulin gene determination. All primer sequences are described in Supplementary Table S1 .
10
Quantitative Real-Time PCR Analysis
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