For quantification of mature miR-125a, specific stem loop primers were used instead of oligo dT to generate specific cDNA of mature miR-125a by using miScript RT kit (Qiagen, Hilden, Germany). The sequence is listed in Supplementary Materials , Table S2 . Expression was analyzed with the miScript SYBR Green PCR kit following the manufacturer’s manual. A QuantStudio™ 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) was used to run the q–PCR and U6were used as an internal control to normalize the expression of miRNA. A 2−ΔΔCT method was used to calculate the fold change (Rao et al., 2013). All the samples were taken in triplicate with three experimental replicates.
Quantstudio 3 real time pcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 3 Real-Time PCR machine is a versatile instrument designed for nucleic acid amplification and detection. It utilizes real-time PCR technology to quantify and analyze DNA and RNA samples. The instrument features a compact design, a high-resolution touchscreen display, and flexible block configurations to accommodate a range of sample volumes and formats.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green master mix, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Taqman rna to ct 1 step kit, by Thermo Fisher Scientific (3 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Ultraplex 1 step toughmix, by Quantabio (2 mentions)
2019 ncov cdc probe and primer kit for sars cov 2, by Quantabio (2 mentions)
Quantstudio design and analysis software version 1, by Thermo Fisher Scientific (2 mentions)
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Quantitect, by Qiagen (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (2 mentions)
Quantitect rt kit, by Qiagen (2 mentions)
Sybr green master mix, by Takara Bio (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
51 protocols using quantstudio 3 real time pcr machine
1
Quantification of Mature miR-125a
2
Quantitative PCR Analysis of iPSN Gene Expression
3
Genotyping Four Key SNPs in Peripheral Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was obtained from peripheral blood withdrawn on EDTA, using commercially available kits (Quick gDNA MiniPrep kit, Zymo Research, USA; PureLink Genomic DNA Mini Kit, Invitrogen, Thermo Fisher, USA). We genotyped four SNPs (FDPS rs2297480, LRP5 rs3736228, SOST rs1234612, VKORC1 rs9934438) in all patients and controls using the real-time PCR technique. Standard, predesigned TaqMan SNP genotyping assays, containing all the primers and probes needed for genotyping, were purchased from Thermo Fisher (codes C___2737970_10, C__25752205_10, C___7566033_10, C__30204875_10). All the genotyping were performed according to manufacturer’s instructions. The reaction mix contained 10 μl of 2xTaqMan Genotyping Master Mix (Applied Biosystems, Thermo Fisher, USA), 0.5 μl of the corresponding 40xTaqMan SNP genotyping assay, approximately 25 ng of genomic DNA and free-nucleases water to the final volume of 20 μl. The same amplification program was used for all the genotyping, consisting in a pre-read stage of 30 seconds (s) at 60°C, hold stage of 10 minutes (min) at 95°C, followed by the PCR stage, consisting of 40 cycles, each comprising 15 s at 95°C and 1 min at 60°C, and a post-read stage of 30 s at 60°C. All the experiments were run on a QuantStudio 3 real-time PCR machine (Applied Biosystems, Thermo Fisher, USA).
4
SARS-CoV-2 Detection and ACE-2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from the cells using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. UltraPlex 1-Step ToughMix (QuantaBio, Beverly, MA, USA) was used along with 2019-nCoV CDC Probe and Primer Kit for SARS-CoV-2 (Catalog: KIT-nCoV-PP1-1000) for the CoV-2 qPCR reactions. Human ACE-2 primer/probe and 18S Ribosomal RNA control (Applied Biosystems, Waltham, MA, USA) were used for the hACE-2 qPCR reactions. QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) was used with QuantStudio Design and Analysis software version 1.5.1 (Applied Biosystems, Waltham, MA, USA) for analysis. Results are expressed as CoV-2 or ACE-2 expression determined using 2−(ΔCt) method with 18S ribosome as the endogenous control.
5
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA and RNA were isolated using a DNA/RNA Isolation Kit (TIANGEN) and reverse transcribed using a miRNA 1st Strand cDNA Synthesis kit (Vazyme). PCR was performed in ChamQ Universal SYBR qPCR Master mix (Vazyme) according to the manufacturer’s instructions in a QuantStudio3 realtime PCR machine (Applied Biosystems). GAPDH mRNA was used to normalize gene transcript levels. The primers used for qPCR and qRT-PCR are listed in S3 Table .
6
Quantitative PCR Analysis of Mouse Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, snap-frozen liver tissue or cultured cells were homogenized in Buffer RLT or RLT + BME and total RNA was isolated from cell lysate using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and complementary DNA was synthesized using the QuantiTect RT Kit (Qiagen) following standard protocols. PCR amplification was performed using either the QuantiTect Sybr green (Qiagen) or TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, CA) PCR kits. Quantitative PCR was performed on a QuantStudio 3 Real-time PCR machine (Applied Biosystems, Waltham, MA) and fold changes in messenger RNA levels were calculated. For each gene, all samples were normalized to the average fold change of the control treatment group (chow, WT, pair-fed, or PBS). The following Qiagen QuantiTect primer assays were used: 18S ribosomal RNA (Rn18s; QT02448075), CD36 (QT01058253); VEGF-D/FIGF (QT QT00164024) for mouse.
7
SARS-CoV-2 RNA Isolation and qRT-PCR Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were scraped and collected in Buffer AVL with carrier RNA (Qiagen, Hilden, Germany). RNA was then isolated from the samples using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. RNA was also isolated from a heat-inactivated cell lysate and supernate containing SARS-CoV-2 isolate USA-WAI/2020 (BEI Resources, Manassas, VA, USA) with known genome equivalents of virus, which was used as a standard during qPCR.
UltraPlex 1-Step ToughMix (QuantaBio, Beverly, MA, USA) was used along with 2019-nCoV CDC Probe and Primer Kit for SARS-CoV-2 (Catalog: KIT-nCoV-PP1-1000) for the qRT-PCR reactions. QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) was used with QuantStudio Design and analysis software version 1.5.1 (Applied Biosystems, Waltham, MA, USA) for analysis. Results are expressed as log of genome equivalents/mL.
UltraPlex 1-Step ToughMix (QuantaBio, Beverly, MA, USA) was used along with 2019-nCoV CDC Probe and Primer Kit for SARS-CoV-2 (Catalog: KIT-nCoV-PP1-1000) for the qRT-PCR reactions. QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) was used with QuantStudio Design and analysis software version 1.5.1 (Applied Biosystems, Waltham, MA, USA) for analysis. Results are expressed as log of genome equivalents/mL.
8
Quantitative Real-time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from at least three silenced plants of each gene was obtained by extraction with RIBOzol Reagent. Remnant genomic DNA was removed by DNase I treatment. First-strand cDNA was synthesized from 0.5 µg of total RNA using RevertAid H Minus Reverse Transcriptase and oligo(dT) (Thermo Fisher Scientific, Carlsbad, CA, USA). Real-time qPCR was carried out using QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) and PyroTaq EvaGreen qPCR Supermix (Solis BioDyne, Tartu, Estonia), specific oligonucleotides, and recommended qPCR cycles as follows: initial denaturation for 12 min at 95°C, followed by 50 cycles of 15 s at 95°C and 60 s at 60°C. Specific oligonucleotides were designed using Primer3web version 4.1.0 (https://bioinfo.ut.ee/primer3 ). Oligonucleotide efficiencies were tested by qRT-PCR using tenfold serial dilutions of the corresponding cDNA. Each biological replicate was run in triplicate. Elongation factor 1-α (EF1α, TC19582), F-BOX family protein (F-BOX, Niben.v0.3.Ctg24993647) and protein phosphatase 2A (PP2A, TC21939) genes were used as endogenous controls (Liu et al., 2012 (link)).
9
Quantification of Glutathione Peroxidase 4 Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated using TRIzol reagent (Invitrogen, Waltham, MA, USA). cDNA synthesis was performed using 1 μg of total RNA and M-MLV Reverse Transcriptase (M1701, Promega). RT-PCR was performed using PCR PreMix (K-2016, BIONEER, Korea)and specific primers [14 ] for 35 cycles. Quantitative RT-PCR was performed using SYBR Green Master Mix (RR420A, Takara Bio, Kusatsu, Shiga, Japan) and specific primers in a QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA). Gene expression was normalized to that of RPL32 mRNA using the 2−ΔCt method. cGPx4 mRNA level was determined by subtracting the 2−ΔCt values of mGPx4 and nGPx4 from that of total GPx4.
Primers sequences were as follows:
GPx4 forward (for RT-PCR), TGT GCG CGC TCC ATG CAC GAG T;
GPx4 reverse (for RT-PCR), AAA TAG TGG GGC AGG TCC TTC TCT;
Total GPx4 forward (for quantitative RT-PCR), GTT TTC CGC CAA GGA CAT CG;
Total GPx4 reverse (for quantitative RT-PCR), ACT TCG GTC TTG CCT CAC TG;
mitochondrial GPx4 forward, ATT GGT CGG CTG GAC GAG;
mitochondrial GPx4 reverse, TCG ATG TCC TTG GCG GAA AA;
nuclear GPx4 forward, CAG CGG TGC CAG AGC C;
nuclear GPx4 forward, GGA CTT GGG ACA TTC GTG GA;
RPL32 forward, CAT CCG GCA CCA GTC AGA CC;
RPL32 reverse, TGT GAG CGA TCT CGG CAC AG.
Primers sequences were as follows:
GPx4 forward (for RT-PCR), TGT GCG CGC TCC ATG CAC GAG T;
GPx4 reverse (for RT-PCR), AAA TAG TGG GGC AGG TCC TTC TCT;
Total GPx4 forward (for quantitative RT-PCR), GTT TTC CGC CAA GGA CAT CG;
Total GPx4 reverse (for quantitative RT-PCR), ACT TCG GTC TTG CCT CAC TG;
mitochondrial GPx4 forward, ATT GGT CGG CTG GAC GAG;
mitochondrial GPx4 reverse, TCG ATG TCC TTG GCG GAA AA;
nuclear GPx4 forward, CAG CGG TGC CAG AGC C;
nuclear GPx4 forward, GGA CTT GGG ACA TTC GTG GA;
RPL32 forward, CAT CCG GCA CCA GTC AGA CC;
RPL32 reverse, TGT GAG CGA TCT CGG CAC AG.
10
Glioblastoma Organoid RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Live cultured glioblastoma organoids underwent one cycle of staining and quenching. After 24 h post cyclic imaging, RNA was extracted from the organoids with Trizol Reagent (ThermoFisher; 15596018), labeled with GlycoBlue Coprecipitant (ThermoFisher; AM9515), and treated with 2U of TURBO DNase (ThermoFisher; AM1907) at 37 °C for 30 min. DNase in activation Reagent was then added to inactivate the TURBO DNase at 0.1 volume for 5 min at RT. The iScript cDNA Synthesis Kit (BioRAD; 1708890) was used for RT‐PCR. With this kit, the cell RNA was mixed with its reagents and run in a SimpliAmp Thermal Cycler (Applied Biosystems) as per the manufacturer's protocol. The qPCR master mix includes PowerTrack SYBR Green Master Mix (Applied Biosystems; A46109), nuclease‐free water, and forward and reverse primers (Integrated DNA Technologies). 10 µL of the qPCR master mix was added to each well. For every qPCR experiment, 40 cycles were run using a QuantStudio 3 Real‐ Time PCR machine (Applied Biosystems). Organoid gene assessment was derived from patients UP‐9121.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!