RNA was isolated from finely crushed snap‐frozen mouse quadriceps and heart samples using Trizol Reagent (Life Technologies), treated with DNAse (Promega; Madison, WI), and reverse transcribed using the SuperScript III kit (Life Technologies). Resulting cDNA was subjected to real‐time PCR using RQG SYBR Green supermix (Qiagen) in a Rotor Gene Q real‐time PCR machine (Qiagen). The following mouse‐specific primers were used: Fbxo32 (forward) 5′‐GCA GCC GCT CAG CAT TCC CA‐3′ and (reverse) 5′‐ACC GAC GGA CGG GAC GGA TT‐3′; Trim63 (forward) 5′‐AGG GCT CCC CAC CAC TGT GT‐3′ and (reverse) 5′‐TTG CCC CTC TCT AGG CCA CCG‐3′; Fbxo30 (forward) 5′‐ATC GAT GGC CCG TTA GTT ATT CA‐3′ and (reverse) 5′‐GCC CCT ATC TCA CCC TCA TCA AG‐3′; Acvr1b (forward) 5′‐GAA CCG CTA CAC AGT GAC CA‐3′ and (reverse) 5′‐ AAT TCC CGG CTT CCC TTG AG‐3′; Tgfbr1 (forward) 5′‐GCA TTG GCA AAG GTC GGT TT‐3′ and (reverse) 5′‐TGC CTC TCG GAA CCA TGA AC‐3′; Tgfb1 (forward) 5′‐GAC TCT CCA CCT GCA AGA CCA T‐3′ and (reverse) 5′‐GGG ACT GGC GAG CCT TAG TTT‐3′; Gapdh (forward) 5′‐AGC AGG CAT CTG AGG GCC CA‐3′ and (reverse) 5′‐ TGT TGG GGG CCG AGT TGG GA‐3′. Relative gene expression quantification was performed using the ΔΔCt method with either Gapdh or Acvr1b as the reference gene.
Rotor gene q real time pcr machine
Manufactured by Qiagen
Sourced in Germany
The Rotor-Gene Q is a real-time PCR machine designed for nucleic acid detection and quantification. It features a rotating sample carousel that can accommodate up to 72 samples simultaneously. The system is capable of performing real-time PCR reactions and provides data analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect primer assay, by Qiagen (3 mentions)
Superscript 3 kit, by Thermo Fisher Scientific (2 mentions)
Rqg sybr green supermix, by Qiagen (2 mentions)
Dnase, by Promega (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Revertra ace cdna synthesis kit, by Toyobo (2 mentions)
Platinum sybr green kit, by Qiagen (2 mentions)
Nucleospin kit, by Macherey-Nagel (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Masterpure yeast dna purification kit, by Illumina (1 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Iscript one step rt pcr kit, by Bio-Rad (1 mentions)
Sybr green master mix kit, by Ampliqon (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
28 protocols using rotor gene q real time pcr machine
1
Gene Expression Profiling in Mouse Tissues
2
Monitoring DNA Double-Strand Break Repair
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Monitoring repair kinetics by qPCR was performed as described previously [50 (link)]. Single colonies were inoculated in 5 ml of media lacking leucine with 2% dextrose and grown overnight at 30°C. Overnight cultures were then diluted into 600 ml of YPLac and grown into log phase. DSBs were induced by adding 20% galactose to a final concentration of 2%. To track the dynamics of DSB repair 50 ml aliquots of each culture was collected every hour over 9 h. DNA was isolated using a MasterPureTM Yeast DNA Purification Kit (Epicentre cat. MPY80200). The repair product, MATa -inc, was amplified using primers MATp13 and MATYp4 with a SYBR Green Master Mix using a Qiagen Rotor-Gene Q real-time PCR machine. To quantify the relative amount of MATa -inc in each sample, SLX4 was used as a reference gene and was amplified using primers NS047-Slx4p7 and Slx4p1. Primer sequences are shown in (S2 Table ).
3
Quantitative Analysis of RNA Levels
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The total RNA from the cell lines, human breast cancer tissues, and para-cancerous tissues used in this study, was extracted with TRIzol reagent and incubated with DNase I (Thermo Fisher, USA) to remove genomic DNA. First-strand complementary DNA (cDNA) was synthesized by using the SuperScript III Reverse Transcriptase Kit (Thermo Fisher, USA). Relative RNA levels determined by RT-qPCR were measured on a Rotor-Gene Q real-time PCR machine (Qiagen, Germany). Glyceraldehyde-3-phosphate dehydrogenase was employed as an internal control. The relative expression of RNAs was calculated using the 2−ΔΔCt method.
4
Real-time RT-PCR for Chikungunya Virus Detection
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The real-time RT-PCR was conducted using the iScript™ One-Step RT-PCR kit (BioRad, Hercules, CA, USA) with the Rotor-Gene Q Real Time PCR machine (Qiagen, Germantown, MD, USA). The samples were assayed in a 25 µL reaction containing 2 µmol/L of forward primer (CHIK/E1/10367/+: 5′-CTCATACCGCATCCGCATCAG-3′), 2 µmol/L of reverse primer (CHIK/E1/10495/-: 5′-ACATTGGCCCCACAATGAATTTG-3′), 5 µL of extracted RNA, 0.25 µL of RNA transcriptase, and 12.5 µL of SYBR® Green Premix. The concentrations of Taq polymerase, buffer, dNTPs, and Mg2+ used were based on the recommendations of the manufacturer. The RT-PCR thermal cycling condition comprised 30 min of reverse transcription step at 50 °C, 15 min of initial denaturation at 95 °C, followed by 40 cycles of amplification steps of denaturation at 95 °C for 30 s, annealing at 55.8 °C for 45 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 10 min. A melting curve was generated after the amplification step at 70–99 °C. A standard curve was constructed by using the synthesized RNA standard with copy numbers ranging from 100 to 1010.
5
Quantifying miRNA-124 and Targets
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Total RNA was extracted using miRNeasy_Mini RNA Extraction Kit (Qiagen, Hamburg, Germany) according to the manufacturer's procedures and stored at −80°C until use. cDNA was synthesized with miScript II RT Kit (Qiagen, Hamburg, Germany). The expression levels of miRNA-124 were determined using the SYBR Green Master Mix Kit (Ampliqon, Odense, Denmark), and the expression levels of potential miRNA-124 targets, STAT-3, SP-1, and SOCS5, were conducted by applying SYBR green Master Mix on a Rotor-Gene Q real-time PCR machine (Qiagen, Hamburg, Germany). The expression levels of STAT-3, SOCS5, and SP1 were normalized to the GAPDH gene, and miR-124 expression was normalized to U6 snRNA as an endogenous control by using 2−ΔΔCt method. The gene-specific primer sets for STAT-3, SOCS5, and SP1 and GAPDH as a housekeeping gene were designed using the NCBI Primer-Blast Tool. The primer mix of U6 and miR-124 was provided from Exiqon (Vedbaek, Denmark). All results are representative of three independent experiments.
6
SARS-CoV-2 Screening and Confirmation Protocol
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According to the recommendations of the Taiwan CDC and WHO guidelines, the SARS-CoV-2 screening and confirmatory assays were performed by targeting the envelope (E) and RNA-dependent RNA polymerase (RdRp) viral genes. In accordance with the protocol suggested by the Taiwan CDC, one-step real-time RT-PCR was performed using the primer and probe sequences designed by Corman et al. [9] (link). The experimental procedure and interpretation of results have been previously described [10] (link), [11] (link). Briefly, one-step real-time RT-PCR was performed on a Rotor-Gene Q real-time PCR machine (Qiagen, Hilden, Germany). Thermal cycling was performed as follows: reverse transcription at 50 °C for 10 min, followed by 95 °C for 2 min, and 50 cycles at 95 °C for 5 s and 58 °C for 30 s. All positive samples were further validated by the Taiwan CDC central laboratory. We evaluated 125 nasopharyngeal swabs (COPAN’s COVID-19 Collection & Transport Kits with Universal Transport Medium or Virus Transport Swabs 147C) from patients suspected of having COVID-19. This study was approved by the Institutional Review Board of the Tri-Service General Hospital (TSGHIRB No. C202005041), registered on March 20, 2020. Informed consent was obtained from patients.
7
FRET-Melting Assay for DNA Structures
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FRET-melting experiments were conducted with FAM-TAMRA dual labeled oligomers listed in Table S1 using a Rotor-Gene Q real-time PCR machine (Qiagen) according to the previous study (De Cian et al., 2007 (link)). Oligonucleotides were tested at 0.25-0.5 μM strand concentration in 50 mM phosphate buffer at different pH. The emission of FAM fluorophore was normalized between 0 and 1, and the melting temperature Tm was determined as the temperature at which the normalized emission equals 0.5.
8
Real-Time qPCR: One-Step SYBR Green
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First strand synthesis and quantitative real-time-PCR amplification were performed in a one-step, single-tube format using the QuantiFastTM SYBR_Green RT-PCR kit from Qiagen according to the manufacturer's protocol in a total volume of 20 μL and gene specific QuantiTect Primer Assays (Qiagen). Thermal cycling and fluorescent detection were performed using the Rotor-Gene Q real-time PCR machine (model 2-Plex HRM) (Qiagen). The SYBR Green I reporter dye signal was measured. Resulting data were analyzed using the HMBS gene as an internal standard to normalize transcript levels. All quantitative real-time PCR reactions were run in technical triplicates.
9
Quantifying Arabidopsis Gene Expression
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Total RNAs from various tissues in Arabidopsis were extracted by using RNA isoPlus reagent (TaKaRa, Japan), and transcribed into cDNA with ReverTra Ace qPCR RT Kit (TOYOBO). The cDNAs were amplified as templates with the gene-specific primers, and quantitative Real-Time PCRs were carried out with a Rotor-Gene Q real-time PCR machine (Qiagen). The transcript abundance relative to WT of different tissues was analyzed by the comparative CT method as described previously (Schmittgen and Livak, 2008 (link); Ma and Zhao, 2010 (link)), and the GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) was utilized as an internal gene for normalizing (Zhong and Simons, 1999 (link)). Three independent biological replicates and three technical replicates of every sample were conducted for quantitative real-time PCR analysis. Primers used in the experiments are listed in Supplementary Table S1 .
10
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was prepared and used to synthesize cDNA using SuperScript II reverse transcriptase, as described (Subramanian et al, 2015 (link)). qRT-PCR was performed using gene-specific primers (Table S1) and QuantiFast SYBR Green PCR Kit in Rotor-Gene Q real-time PCR machine (QIAGEN) and ViiA7 real-time PCR system (Thermo Fisher Scientific) according to manufacturer’s instructions, and relative gene expression was normalized with Gapdh expression. 2−∆∆Ct was used to compare the transcriptional differences. Cycle threshold (Ct) values of various genes were plotted against Ct values of Gapdh to determine a ratio between different treatment groups.
Table S1
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