To confirm the RNA‐Seq expression of the significant DEGs analyses, total RNAs were extracted from aerial and submerged mycelia following 5, 12 and 19 days of culture. The quantitative RT‐PCR was quantified using RNA‐DirectTM SYBR green real‐time PCR master mix (Toyobo Co., LTD, Osaka, Japan) using the following conditions: 90°C: 30 s, 60°C: 20 min, 95°C: 1 min, 95°C: 15 s; 59°C: 15 s, and 74°C: 50 s for 40 cycles and performed in Mx3000P QPCR. Nine genes were selected and quantified using quantitative RT‐PCR (qRT‐PCR), including alcohol dehydrogenase CCM_09633, alcohol dehydrogenase CCM_02484, hexokinase CCM_06280, aldehyde dehydrogenase CCM_02203, glucose‐6‐phosphate dehydrogenase CCM_06983, adenylate cyclase, putative CCM_02396, oxidoreductase CCM_ 01934, succinate dehydrogenase CCM_07146, and 5’‐nucleotidase CCM_00622. The primer sets used are listed in Appendix Table A2 . The relative gene expression was calculated using the 2−ΔΔCT method (Livak & Schmittgen, 2001 ), and the Rho GTPase activator (Sac7) CCM_07283 (Llanos, Francois, & Parrou, 2015 ) was used as a reference gene to quantify the relative expression levels of the nine genes.
Rna direct sybr green realtime pcr master mix
Manufactured by Toyobo
Sourced in Japan, United States
The RNA-direct SYBR Green Realtime PCR Master Mix is a laboratory reagent designed for real-time quantitative reverse transcription PCR (RT-qPCR) analysis. It contains all the necessary components for the amplification and detection of RNA targets, including a SYBR Green-based fluorescent dye for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (4 mentions)
Thunderbird sybr qpcr mix, by Toyobo (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Revertra ace qpcr rt kit, by Toyobo (4 mentions)
Itaq sybr green supermix, by Bio-Rad (2 mentions)
Abi 7900ht qrt pcr system, by Thermo Fisher Scientific (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Toyobo (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
High pure pcr template preparation kit, by Roche (2 mentions)
Mx3005p real time qpcr system, by Agilent Technologies (1 mentions)
Nucleospin rna column, by Takara Bio (1 mentions)
Thermal cycler dice real time system 3, by Takara Bio (1 mentions)
Cellamp direct rna prep kit for rt pcr, by Takara Bio (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Ifn αa d, by Merck Group (1 mentions)
Isogen 2 reagent, by Nippon Gene (1 mentions)
20 protocols using rna direct sybr green realtime pcr master mix
1
Quantitative Validation of RNA-Seq DEGs
2
Quantification of Viral RNA Levels
3
Quantification of Viral RNA Levels
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Total RNA extraction was performed with the RNeasy mini Kit (Qiagen). Detection of HAV genome RNA was carried out by a two-step quantitative RT-PCR analysis with the SuperScript®III First-Strand Synthesis System (Thermo Fisher Scientific) and iTaq SYBR Green Supermix (Bio-Rad) or alternatively Thunderbird SYBR qPCR Mix (TOYOBO) using specific primers 5’-GGTAGGCTACGGGTGAAAC-3’ and 5’-AACAACTCACCAATATCCGC-3’. HCV RNA level was determined as previously described32 (link). Quantification of IRF1 target genes was performed with the primer pairs listed in Supplementary Table 3 . DENV and ZIKV RNA levels were quantified using specific primer pairs targeting DENV genome RNA, 5’-ACACCACAGAGTTCCATTACAGA-3’ and 5’-CATCTCATTAAAGTCGAGGCC-3’, or ZIKV genome RNA, 5’-AARTACACATACCARAACAAAGTGGT-3’ and 5’- TCCRCTCCCYCTYTGGTCTTG-3’ respectively, using RNA-directTM SYBR Green Realtime PCR Master Mix (TOYOBO).
4
Quantitative RT-PCR Analysis of HCC
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Total RNA was extracted from HCC samples and cell lines using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by a spectrophotometer (NanoDrop™ ND-1000). Total RNA (1 μg) was reversely transcribed using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). QRT-PCR was performed using RNA-direct TMSYBR® GreenReal-time PCR Master Mix (Toyobo, Osaka, Japan). The amplified primers were shown in Table 1. Relative level was measured with the ABI 7900HT qRT-PCR system (Applied Biosystems, Foster City, CA, USA) and calculated by 2 -ΔΔCt method.
5
Quantitative Analysis of Growth Factors in Rat Wound Tissues
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Rat tissue was added to 1 ml of PBS and homogenised. RNA was isolated from 500 µl of homogenate by using the Nucleo-pore® RNASure® Mini Kit (Genetix: NP-84105) and used in a quantitative real-time PCR assay with the RNA-direct™ SYBR® Green Realtime PCR Master Mix (Toyobo: QRT-201). The q-RT-PCR was done on a QuantStudio™ 3 Real-Time PCR System.
Expression levels of TGF-ß1, VEGF, and EGF in wound tissues of rats in each group was also estimated using pooled samples per group by one-step SYBR Green Quantitative Real-time PCR (qRT-PCR) (to see relative quantification by fold change) to confirm the ELISA assay results.
Expression levels of TGF-ß1, VEGF, and EGF in wound tissues of rats in each group was also estimated using pooled samples per group by one-step SYBR Green Quantitative Real-time PCR (qRT-PCR) (to see relative quantification by fold change) to confirm the ELISA assay results.
6
Quantitative RT-PCR Analysis of M. capsulatus
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Total RNA was isolated from M. capsulatus (Bath) cells by using a bead-beating method as described previously (Kato et al., 2014 (link)). The PCR primers used for quantitative reverse transcription (qRT)-PCR were designed with Primer3 software (Untergasser et al., 2012 (link)) and are listed in Supplementary Table S1 . Quantitative expression analysis based on one-step real-time RT-PCR was performed using RNA-direct SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) and the Mx3000P System (Stratagene, San Diego, CA, United States) as described previously (Kato et al., 2014 (link)). The gene for outer membrane protein B (mopB), a housekeeping gene of M. capsulatus (Bath) (Karlsen et al., 2005 (link)), was used to normalize expression values.
7
Transcriptional Profiling of Metabolic Genes
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The transcriptional expression of maeB, ppsA, ppc, pckA, pgi, zwf, gltA, and arcA in each strain was quantified using real-time PCR. Total RNA was isolated from individual cultures using a NucleoSpin RNA column (Takara Bio, Shiga, Japan) according to the manufacturer's protocol. Reverse transcription reactions and quantitative real-time PCR was performed using an Mx3005P Real-Time QPCR System (Agilent Technologies, Santa Clara, CA, USA) with RNA-direct SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan). The primer pairs are listed in Supplementary Data 7 . The normalized transcriptional level of each mRNA was calculated by the relative quantification method using the mdoG gene (coding glucan biosynthesis protein G) as the housekeeping gene61 (link).
8
Quantifying Replicon Genomes in EV Samples
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Total RNAs were obtained by CellAmp™ Direct RNA Prep Kit for RT-PCR (TaKaRa Bio Inc., Shiga, Japan) from C6/36 cells in a 96-well plate inoculated with EVs derived from BHK/JM-PnL and BHK/DN-PnL cells. Total RNAs of the EVs treated with RNase A in the presence/absence of Tx-100 were isolated by a microRNA Extractor® Kit for Purified EV (FUJIFILM Wako Pure Chemical Corporation). JM-PnL and DN-PnL replicon genomes were quantified by RNA-direct™ SYBR® Green Realtime PCR Master Mix (TOYOBO). Primers used for JM-PnL were sense, 5′-ccctcagaaccgtctcggaa-3′, and anti-sense, 5′-ctattcccaggtgtcaatatgctgt-3′, and primers used for DN-PnL were sense, 5′-agttgttagtctacgtggaccga-3′, and anti-sense, 5′-cgcgtttcagcatattgaaag-3′. The real-time PCR was carried out by Thermal Cycler Dice Real-Time System III (Takara Bio Inc.). The in vitro-transcribed JM-PnL and DN-PnL were used as standard.
9
Quantifying AEBP1 Expression in Glioblastoma
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The relative mRNA expression of AEBP1 was evaluated using qRT-PCR. Total RNA was extracted from GBM tissues and cell lines using the TRIzol reagent (Invitrogen). RNA was reversely transcribed into cDNA using a reverse transcription kit (Takara, Japan). PCR reactions were performed with the RNA-Direct SYBR Green Real-Time PCR Master Mix (Toyobo, Japan) and Roche LightCycler 480 Real-Time PCR System (Applied Biosystems, USA). The relative expression of genes was calculated by the 2-ΔΔCt method. The primers used for the experiment were as follows: AEBP1, forward: 5′-ACCCACACTGGACTACAATGA-3′ and reverse: 5′-GTTGGGGATCACGTAACCATC-3′, and GAPDH used as the internal reference, forward: 5′-TATGATGATATCAAGAGGGTAGT-3′ and reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′.
10
Quantification of MuV Genome and RIG-I mRNA
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Total RNA was extracted from the cells and culture supernatants using a RNeasy Mini Kit (Qiagen, Germantown, MD, United States) according to the manufacturer’s protocol. MuV genome quantification has been described previously (Katoh et al., 2017 (link)). For RIG-I mRNA quantification, A549/hSLAM cells were treated with 0–10 μM of CD437 and 1,000 Units/ml of IFN αA/D (Sigma-Aldrich) for 24 h. Total RNA was extracted and one-step quantitative RT-PCR was performed using the RNA-direct SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) in a LightCycler 480 system using the following primers: 5′-CTTTTTCTCAAGTTCCTGTTGGA-3′ and 5′-TCCCAACTTTCAATGGCTTC-3′ (Hergovits et al., 2017 (link)). RNA expression was normalized to that of hypoxanthine phosphoribosyltransferase 1 using the following primers: 5′-CATTATGCTGAGGATTTGGAAAGG-3′ and 5′-CTTGAGCACACAGAGGGTACA-3′.
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