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Quantstudio 3 real time pcr

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The QuantStudio 3 Real-Time PCR System is a laboratory instrument used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is designed to perform real-time detection and quantification of nucleic acid targets, such as DNA and RNA, in biological samples.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (4 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions) Rneasy mini kit, by Qiagen (3 mentions) Sybr green master mix, by Thermo Fisher Scientific (3 mentions) Steponeplus, by Thermo Fisher Scientific (2 mentions) Power sybr green pcr master mix reagent, by Thermo Fisher Scientific (2 mentions) Quantstudio design analysis software v1, by Thermo Fisher Scientific (2 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Protein thermal shift dye, by Thermo Fisher Scientific (2 mentions) Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Iscript cdna synthesis kit, by Bio-Rad (2 mentions) Protein thermal shift software, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Power sybr green master mix, by Thermo Fisher Scientific (1 mentions) High capacity cdna reverse transciption kit, by Thermo Fisher Scientific (1 mentions) Miniamp plus thermal cycler, by Thermo Fisher Scientific (1 mentions) Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)

33 protocols using quantstudio 3 real time pcr

1

Quantification of mRNA and miRNA Levels

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Total RNA content from cells and tissue were isolated using TRIzol reagent, and cDNA was synthesized using a iScript cDNA Synthesis kit (Bio-Rad, CA, USA). mRNA levels of candidate genes were quantified with qPCR (QuantStudio-3 real time PCR, LifeTechnologies, Carlsbad, CA, USA) using a SYBR Select Master Mix. The data were normalized with the internal control (18S or GAPDH) and analyzed using the 2−ΔΔCT method. The miRNeasy Kit (Qiagen, Germany) was used for total miRNA isolation, and cDNA was synthesized using a miScript II RT kit (Qiagen, Germany). The levels of miRNA were quantified with qPCR (QuantStudio-3 real-time PCR, LifeTechnologies, CA, USA) using a SYBR Select Master Mix, and data were normalized to the internal control 5S and analyzed using the 2−ΔΔCT method. A reaction tube w/o a template was used as a negative control, and all the samples were run with n = 3 technical replicates. Primer details are listed in Supplementary Tables S1 and S2.
Vyas H.S., Jadeja R.N., Vohra A., Upadhyay K.K., Thounaojam M.C., Bartoli M, & Devkar R.V. (2023). CORM-A1 Alleviates Pro-Atherogenic Manifestations via miR-34a-5p Downregulation and an Improved Mitochondrial Function. Antioxidants, 12(5), 997.
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2

Quantitative RT-PCR for Gene Expression

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One microgram of total RNA was reverse transcribed to cDNA [37 (link)] and amplified with gene-specific primers labeled with Power SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) using an QuantStudio 3 Real time PCR (Applied Biosystems). Samples were normalized to the expression of 18S rRNA using the comparative CT method [82 (link)]. Sequences for the primers (Il1b, Tlr4, Nalp3) can be found in Table S1 or in previously published works (Kc, Mcp1, Cox2, Il6, 18S) [36 (link),37 (link),40 (link)].
Romo D., Velmurugan K., Upham B.L., Dwyer-Nield L.D, & Bauer A.K. (2019). Dysregulation of Gap Junction Function and Cytokine Production in Response to Non-Genotoxic Polycyclic Aromatic Hydrocarbons in an In Vitro Lung Cell Model. Cancers, 11(4), 572.
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3

Quantitative RT-PCR Analysis of HTLA and HUVECs

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For real-time PCR (RT-PCR), HTLA and HUVECs underwent the following protocol. RNA isolation was conducted using the RNeasy Mini Kit (74104, Qiagen, Hilden, Germany), followed by the quantification of the isolated product using the NanoDrop One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Fisher Scientific, Grand Island, NY). cDNA was generated using the High-Capacity cDNA Reverse Transciption Kit (4368814, Applied Biosystems, Grand Island, NY) using the Miniamp Plus Thermal Cycler (Applied Biosystems). RT-PCR was performed using Fast SYBR Green Master Mix (4385610, Applied Biosystems) and the QuantStudio 3 Real-Time PCR System (Applied Biosystems) using the following primers and conditions:
QuantStudio 3 Real-Time PCR Conditions:
RT-PCR Primers:
Li K.J, & Garoff H. (1998). Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors. Proceedings of the National Academy of Sciences of the United States of America, 95(7), 3650-3654.
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4

Quantitative miRNA Binding Assay

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All 72 microwells on each of the 5 electrodes of the chip were immobilized with P155 probes. Different electrochemical redox binding (EREB) condition was applied on each electrode with fresh solution. The difference in miR-155 target copy number in a 500-μL solution of EREB before and after binding was measured by qPCR (QuantStudio 3 Real Time PCR, Applied Biosystems) to obtain target molecule coverage, NT. The details of the qPCR method and the standard curve are described in the SI, Section S3.
Subsequently, the Au–S linkage was electrochemically cleaved by applying a CV ramp from −1.0 to +0.5 V in 50 mM K4[Fe(CN)6]. The solution was analyzed by qPCR to obtain the copy number of the cleaved probe (NP). From the binding fraction, f = NT/NP as a function of EREB conditions was obtained for each electrode. The NP was also measured by fluorescence (SI, Section S6).
Tevatia R., Prasad A, & Saraf R.F. (2019). Electrochemical Characteristics of a DNA Modified Electrode as a Function of Percent Binding. Analytical chemistry, 91(16), 10501-10508.
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5

RNA Extraction and Quantitative PCR

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Mouse tissues were sectioned in 30‐μm thin slices, collected in 1.4 mm Zirconium Beads pre‐filled tubes (OPS Diagnostic), and homogenized using a MagNA Lyser (Roche Diagnostic). RNA extraction was performed using Trizol reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Next, 1 μg of RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific) following the manufacturer's protocol.
Quantitative PCR was performed using the fast PowerUp SYBR Green Master mix (Thermo Fisher) on a QuantStudio3 real‐time PCR (Applied Biosystems). DNA and RNA editing were analyzed with the primers reported in Appendix Table S6. ΔΔCt was analyzed to assess fold changes between edited and unedited samples.
Maino E., Wojtal D., Evagelou S.L., Farheen A., Wong T.W., Lindsay K., Scott O., Rizvi S.Z., Hyatt E., Rok M., Visuvanathan S., Chiodo A., Schneeweiss M., Ivakine E.A, & Cohn R.D. (2021). Targeted genome editing in vivo corrects a Dmd duplication restoring wild‐type dystrophin expression. EMBO Molecular Medicine, 13(5), e13228.
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6

Aptamer Library Complexity Evaluation

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Aptamer library complexity was evaluated as described previously38 (link),94 (link) using 0.5 mM dsDNA from rounds 0, 3, 6, 7, 8, and 9 in triplicate. Samples were combined with iQ SYBR Green Supermix (Bio-Rad, 1708880) at a 1:1 volume and subjected to a reverse DNA melt curve protocol (95°C–25°C, ramp over 20 min) using a quantitative PCR machine (Applied Biosystems QuantStudio 3 Real-Time PCR). Data were plotted as fluorescence (SYBR Green intensity) against increasing temperature (°C).
Ruiz-Ciancio D., Lin L.H., Veeramani S., Barros M.N., Sanchez D., Di Bartolo A.L., Masone D., Giangrande P.H., Mestre M.B, & Thiel W.H. (2023). Selection of a novel cell-internalizing RNA aptamer specific for CD22 antigen in B cell acute lymphoblastic leukemia. Molecular Therapy. Nucleic Acids, 33, 698-712.
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7

RNA Extraction and Gene Expression Analysis of Human Pancreatic Islets

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Extraction of total RNA from intact human pancreatic islets was performed using the RNeasy Plus Mini Kit (Qiagen, Germany). RNA quantity and quality were measured by NanoDrop 2000 and Agilent 2100 Bioanalyzer. For cDNA synthesis, a high-capacity cDNA synthesis kit was used (Thermo Fisher Scientific, USA). Silencing efficiency and gene expression analysis were assessed using TaqMan gene expression assays; PLCXD3 (Rn01762608_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), MAFA (Rn00845206_g1), PDX1 (Rn00755591_m1), INSR (Rn00690703_m1), GCK (Rn00561265_m1), GLUT2 (Rn00563565_m1). HPRT1 (Rn01527840_m1) was used as an endogenous control to normalize the expression of target mRNA. SYBR Green qPCR gene expression analysis with the corresponding primers (Table 1) was used for NEUROD1, NKX2.2, and HPRT1. Relative gene expression was performed using the 2−ΔΔCt method. All reactions were executed in QuantStudio 3 Real-time PCR (Applied Biosystems, USA).
Aljaibeji H., Mukhopadhyay D., Mohammed A.K., Dhaiban S., Hachim M.Y., Elemam N.M., Sulaiman N., Salehi A, & Taneera J. (2019). Reduced Expression of PLCXD3 Associates With Disruption of Glucose Sensing and Insulin Signaling in Pancreatic β-Cells. Frontiers in Endocrinology, 10, 735.
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8

Quantitative mRNA and miRNA Analysis

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Quantitative RT-PCR analyses of mRNA (SYBR Green) and miRNA (Taqman) levels were performed according to manufacturer’s instructions with the StepOnePlus and QuantStudio 3 Real-Time PCR Systems (Applied Biosystems). Levels were normalized to Y45F10D.4 for mRNAs and U18 snoRNA for miRNAs.
Aalto A.P., Nicastro I.A., Broughton J.P., Chipman L.B., Schreiner W.P., Chen J.S, & Pasquinelli A.E. (2018). Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging. PLoS Genetics, 14(6), e1007379.
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9

Thermal Stability Analysis of K203.A Antibody

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A protein thermal shift assay was conducted to investigate the thermal stability of the K203.A antibody. In each well of a MicroAmp® Optical 8-tube strip (Applied Biosystems, Foster City, CA, USA), 5 μg of K203.A antibody was added. Additionally, 2.5 μL of Protein Thermal Shift Dye (8×, Applied Biosystems, Foster City, CA, USA) was added. For the negative control, PBS was mixed with Protein Thermal Shift Dye. The thermal shift measurements were performed using a real-time PCR instrument (QuantStudio™ 3 Real-Time PCR, Applied Biosystems), according to the manufacturer’s instructions. Furthermore, all experiments were performed in duplicate to ensure reliability and accuracy.
Kim J.W., Kim H.J., Heo K., Lee Y., Jang H.J., Lee H.Y., Park J.W., Cho Y.B., Lee J.H., Shin H.G., Yang H.R., Choi H.L., Shim H.B, & Lee S. (2023). A novel bispecific antibody dual-targeting approach for enhanced neutralization against fast-evolving SARS-CoV-2 variants. Frontiers in Immunology, 14, 1271508.
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10

Quantitative Analysis of JAK2 mRNA Expression

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The mRNA expression levels of JAK2 were analyzed by qRT-PCR with QuantStudio® 3 Real-Time PCR (Applied Biosystems). The gene-specific primers used for qRT-PCR are listed in Table 1. C. Idellaβ-actin was used as an internal control to normalize the samples. Real-time PCR was performed in triplicate for each complementary DNA sample using SYBR Green I in a final volume of 12.5 μL (6 μL of SYBR® Premix Ex Taq (2×), 0.5 μL cDNA, 0.5 μL of each primer (10 μM) and 5 μL of ddH2O). The PCR conditions were as follows: 95°C for 30 min followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. To maintain consistency, the baseline was set automatically by the software. The relative mRNA expression level was analyzed using the comparative CT method [2−ΔΔCt (ΔΔCt=(Ct gene  of interest−Ct  β-actin)treat− (Ct gene  of interest−Ct  β-actin)untreat)]  with CFX Manager software (Chaoyue et al., 2019 (link)).
Luo W.J., Song P., He Z.M., Cao S.P., Tang J.Z., Xu W.Q., Xiong D., Qu F.F., Zhao D.F., Liu Z., Li J.Z, & Yin Y.L. (2020). JAK2 Mediates the Regulation of Pept1 Expression by Leptin in the Grass Carp (Ctenopharyngodon idella) Intestine. Frontiers in Physiology, 11, 79.
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