RNA was isolated using Trizol (PeqGOLD TriFast, Peqlab, VWR, Vienna, Austria) followed by purification with the E.Z.N.A. Microelute Total RNA Kit (Omega Bio-Tek, VWR, Vienna, Austria), including the optional DNA digestion step (RNase-free DNase I Set, Omega Bio-Tek, VWR, Vienna, Austria). After quantification equal amounts of total RNA were reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For RT-qPCR, cDNA and gene specific primers were used with the Maxima® SYBR Green/ROX RT-qPCR MM (2X) (Fermentas Fisher Scientific, Austria) or the GoTaq RT-qPCR Master Mix (Promega, Mannheim, Germany). Samples were normalized to the geometric mean of two reference genes (gapdh, rplp0). Oligonucleotide primer sequences were: L1-RNP-ORF1_fwd-AGGAAAGCCCATCAGACTAACAG; L1-RNP-ORF1_rev-GGCCTGGTGGTGACAAAATCT; L1-RNP ORF2-s-GAAATGGATACATTCCTCGACACA; L1-RNP-ORF2-as-CTGGTCCTGGACTCTTTTTGGT; GAPDH_f-TGCACCACCAACTGCTTAGC; GAPDH_r-GGCATGGACTGTGGTCATGAG; RPLP0_f-AGCCCAGAACACTGGTCTC; and RPLP0_r-ACTCAGGATTTCAATGGTGCC; APOBEC3C_f-GCATATCTAAGAGGCTGAACATGAAT; APOBEC3C_r-TGGAAGTAGAATGTGCCTGGATAC; APOBEC3H_f-CAGCTGACGCCGCAGAAT; APOBEC3H_r-GACTTGATCTCGTTAATAAAGCAAATTTC; APOBEC3G_f-GTGGAGCGCATGCACAATG; APOBEC3G_r-GGCCTTCAAGGAAACCGTGT; ACTB_f- AGGCACCAGGGCGTGAT; ACTB_r-TGTAGAAGGTGTGGTGCCAGATT; TOP3A_f-GCATCGACTCTTTAACCACACGG; or TOP3A_r-CTCCACAGTGTTCCAAGGCTTGA.
Gotaq rt qpcr master mix
Manufactured by Promega
Sourced in Austria, United States, Germany
GoTaq RT-qPCR Master Mix is a ready-to-use solution for reverse transcription and real-time quantitative PCR (RT-qPCR) reactions. It contains all the necessary components, including the GoTaq DNA Polymerase, reverse transcriptase, and buffers, to perform both the reverse transcription and qPCR steps in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Peqgold trifast, by Avantor (3 mentions)
Microelute total rna kit, by Omega Bio-Tek (3 mentions)
Rnase free dnase 1 set, by Omega Bio-Tek (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Ready to go you prime first strand beads, by GE Healthcare (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Trizol, by Merck Group (1 mentions)
7500 real time pcr system, by Promega (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
8 protocols using gotaq rt qpcr master mix
1
RNA Extraction and RT-qPCR for L1-ORF and APOBEC Gene Expression
2
RT-qPCR for Immune Gene Expression
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Reverse transcription-quantitative PCR (RT-qPCR) analysis was adopted to validate immunity-related gene expression patterns from RNA-seq results. RNA samples were prepared using the same method we described above. A total of 2 μg of RNA was reverse transcribed using PrimeScriptTM RT Master Mix (Takara, Shiga, Japan) and RT-qPCR was performed on an ABI 7300 Real-Time PCR System (United States) using GoTaq RT-qPCR Master Mix (Promega, Madison, WI, United States). All samples obtained at different timepoints have corresponding Tween controls. Three biological replications were conducted for each treatment. Four technical replications were done for each sample in RT-qPCR. PCR conditions consisted of 94°C for 5 s, followed by 40 cycles of 59°C for 20 s and 72°C for 20 s. The primers used are described in Supplementary Table 1 . The RPS15 gene was used as the reference gene (Luo et al., 2018 ; Lü et al., 2018 (link)). Data were collected, exported to EXCEL, and analyzed by the 2–ΔΔCt method (Schmittgen and Livak, 2008 (link)). The mean Ct value of four technical replications in each sample was used for the gene expression analysis, and the relative expression level of each treatment was the average of the three biological replications.
3
Quantitative Gene Expression Analysis
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Total RNA was isolated using TRI Reagent (Sigma-Aldrich), complementary DNA was synthesized using Ready-To-Go-You-Prime-First-Strand Beads (GE Healthcare), and RT-qPCR used GoTaq RT-qPCR Master Mix (Promega) and Eppendorf fluorescence thermocyclers, all according to manufacturers’ instructions. The 2ΔΔCT method was used to quantify amplified fragments. Expression levels were normalized using at least one housekeeping gene (gapdh and actin). Primer sequences are listed in Table S2.
4
Quantification of CD44 Expression by RT-qPCR
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Total RNA was isolated using Trizol (Sigma-Aldrich, Taufkirchen, Germany), complementary DNA was synthesized using Ready-To-Go-You-Prime-First-Strand Beads (GE Healthcare (VWR), Bruchsal, Germany) and RT-qPCR performed using GoTaq RT-qPCR Master Mix (Promega, Mannheim, Germany) and Eppendorf fluorescence thermocyclers (Eppendorf, Wesseling-Berzdorf, Germany), all following manufacturers' instructions. The 2ΔΔCT method was used to quantify amplified fragments. Expression levels were normalized using at least one housekeeping gene (rps29 and/or rpl4). The sequence of the qPCR primers can be found in ref. 47 (link). The sequences of the primers used for CD44 are: 5′-GCACTGTGACTCATGGATCC-3′ present in exon 16 and 5′-GCACTGTGACTCATGGATCC-3′ in exons 17–18.
5
Quantitative Real-Time PCR Analysis
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Cells total RNA was extracted with TRIzol reagent (Vazyme, Nanjing, China). 1 ug of RNA was reverse transcribed to cDNA using the GoScript Reverse Transcription System (Promega, USA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out by the Applied Biosystems 7500 Real-Time PCR System using the GoTaq RT-qPCR Master Mix (Promega, A6001). We chose GAPDH to normalize TFAP2A and PD-L1 expression levels. Relative gene expression was calculated using the 2-ΔΔCt method. All the sequences of qPCR primers were shown in Supplementary Table 1 .
6
RNA Isolation and qRT-PCR for mRNA and miRNA
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For mRNA of all samples, RNA was isolated using Trizol (PeqGOLD TriFast, Peqlab, VWR, Vienna, Austria) followed by purification with the E.Z.N.A. Microelute Total RNA Kit (Omega Bio-Tek, VWR, Vienna, Austria), including the optional DNA digestion step (RNase-free DNase I Set, Omega Bio-Tek, VWR, Vienna, Austria) according to manufactures’ instructions. For RT-qPCR, RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany), followed by qPCR with the GoTaq RT-qPCR Master Mix (Promega, Mannheim, Germany) according to manufacturer’s instructions.
For miRNA measurements, RNA was isolated from exosome pellet with miRNeasy kit (Quiagen, Hilden, Germany) according to manufacturer’s instruction. cDNA was generated with the miScript II RT Kit and was used as a template for real-time PCR with the miScript SYBR Green PCR Kit (Quiagen, Hilden, Germany) in accordance with the manufacturer’s protocol and a gene-specific probe in a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative expression level for each miRNA was computed using the comparative CT method [31 (link)]. miRNA expression was normalized to small nucleolar RNA U6. For mRNA analysis, samples were normalized to the geometric mean of two reference genes (GAPDH, RPLP0). Primer sequences are listed inSupplement Table S2 .
For miRNA measurements, RNA was isolated from exosome pellet with miRNeasy kit (Quiagen, Hilden, Germany) according to manufacturer’s instruction. cDNA was generated with the miScript II RT Kit and was used as a template for real-time PCR with the miScript SYBR Green PCR Kit (Quiagen, Hilden, Germany) in accordance with the manufacturer’s protocol and a gene-specific probe in a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative expression level for each miRNA was computed using the comparative CT method [31 (link)]. miRNA expression was normalized to small nucleolar RNA U6. For mRNA analysis, samples were normalized to the geometric mean of two reference genes (GAPDH, RPLP0). Primer sequences are listed in
7
Quantifying Gene Expression by qPCR
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Gene expression was analyzed by quantitative polymerase chain reaction (qPCR), and the primers used in this study (Table 1 ) were designed using the Primer3 online tool (https://primer3.ut.ee/http://bioinfo.ut.ee/primer3–0.4.0/ ) and the sequences of the identified fragments. The qPCR assay was performed using CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, United States) and GoTaq® RT-qPCR Master Mix (Promega, United States). The amplification conditions were 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 60 s, followed by the requirements needed to calculate the melting curve. The 2–ΔΔCT method was used to normalize the fold change in gene expression using actb as a reference gene for normalization (Yang et al., 2013 (link)).
8
Isolation and Quantification of Aortic Cells
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RNA isolation and RT‐qPCR were performed in isolated and CD34+/c‐kit+ sorted aortic TCs. RNA was isolated using TRIzol (PeqGOLD TriFast, Peqlab, VWR, Vienna, Austria) followed by purification with the E.Z.N.A. Microelute Total RNA Kit (Omega Bio‐Tek, VWR, Vienna, Austria), including the optional DNA digestion step (RNase‐free DNase I Set, Omega Bio‐Tek, VWR, Vienna, Austria) according to manufactures’ instructions. RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For RT‐qPCR, cDNA and gene‐specific primers were used with the Maxima® SYBR Green/ROX RT‐qPCR MM (2X) (Fermentas, Fisher Scientific, Austria) or the GoTaq RT‐qPCR Master Mix (Promega, Mannheim, Germany). Samples were normalized to the geometric mean of two reference genes (GAPDH, RPLP0). Primer sequences are listed in Table 2 .
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