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Itaq universal sybr green kit

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The ITaq Universal SYBR Green kit is a real-time PCR reagent designed for the detection and quantification of DNA sequences. It contains all the necessary components for the amplification and detection of target DNA using the SYBR Green I dye.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (11 mentions) Iscript cdna synthesis kit, by Bio-Rad (6 mentions) Rneasy kit, by Qiagen (3 mentions) Iscript complementary dna cdna synthesis kit, by Bio-Rad (3 mentions) Cfx96 instrument, by Bio-Rad (3 mentions) Reverse transcription kit, by Thermo Fisher Scientific (3 mentions) Rnaeasy kit, by Qiagen (2 mentions) Iscript kit, by Bio-Rad (2 mentions) Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions) Primerquest tool, by Integrated DNA Technologies (2 mentions) Quantistudio 6 flex system, by Thermo Fisher Scientific (2 mentions) Quantistudio real time pcr softwarev1, by Thermo Fisher Scientific (2 mentions) Primescript rt reagent kit, by Takara Bio (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions) Stratagene mx3005p, by Agilent Technologies (1 mentions) Rnase free dnase set, by Qiagen (1 mentions) Iscript reverse transcription supermix for rt qpcr, by Bio-Rad (1 mentions) E z n a hp total rna kit, by Omega Bio-Tek (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Superscript 3 first strand kit, by Thermo Fisher Scientific (1 mentions)

22 protocols using itaq universal sybr green kit

1

Quantitative Real-Time PCR Analysis

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Total RNA was purified at indicated times with the RNAeasy kit (Qiagen, Germantown, MD, USA). In all, 1 μg of purified RNA was DNase digested for 1 h at 37 °C (Ambion, Burlington, ON, Canada) and cDNA was synthesized using the iScript kit (Bio-Rad, Mississauga, ON, Canada). cDNA was PCR amplified on a Stratagene MX3005p real-time thermocycler (Agilent Technologies, Mississauga, Ontario, Canada) using a iTaq universal SYBR Green kit (Bio-Rad). Relative transcript expression was computed using the ΔΔCt method.
Marchildon F., Fu D., Lala-Tabbert N, & Wiper-Bergeron N. (2016). CCAAT/enhancer binding protein beta protects muscle satellite cells from apoptosis after injury and in cancer cachexia. Cell Death & Disease, 7(2), e2109-.
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2

Quantifying RNA Expression in Liver Tissue

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TRIzol reagent (Thermo Fisher Scientific, Inc.) was applied for the separation of total RNA from liver tissue or cells. cDNA was generated by using a Reverse Transcription Kit (Thermo Fisher Scientific, Inc.). And the iTaq Universal SYBR Green kit (Bio-Rad Laboratories, Inc. CA, USA) was then used to perform RT-qPCR. The study was performed with GAPDH as the internal reference gene. The 2−ΔΔCq method was for relative quantification. The primer sequences used were as follows: colla1, mouse forward 5’-GGGGCAAGACAGTCATCGAA −3’ and reverse 5’-GAAGTAGACGGGGTTGAGGG −3’, human forward 5’-TGACGAGACCAAGAACTGCC-3’ and reverse 5’-GCACCATCATTTCCACGAGC −3’; α-SMA, mouse forward 5’-TGACTGAGCGTGGCTATTCC-3’ and reverse 5’-GCCAGGGCTACAAGTTAAGG −3’, human forward 5’-CCGGGACTAAGACGGGAATC-3’ and reverse 5’-ATGGGGACATTGTGGGTGAC −3’; GAPDH, mouse forward 5’-AGGTCGGTGTGAACGGATTTG-3’ and reverse 5’-GGGGTCGTTGATGGCAACA −3’, human forward 5’-CTGGGCTACACTGAGCACC-3’ and reverse 5’-AAGTGGTCGTTGAGGGCAATG −3’.
Ding W., Zhou D., Zhang S., Qian J., Yang L, & Tang L. (2022). Trimetazidine inhibits liver fibrosis and hepatic stellate cell proliferation and blocks transforming growth factor-β (TGFβ)/Smad signaling in vitro and in vivo. Bioengineered, 13(3), 7147-7156.
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3

Quantitative Real-Time PCR for Gene Expression

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Total RNA from the cell pellets was extracted using RNeasy Kit (Qiagen) with the removal of genomic DNA using the RNase-free DNase Set (Qiagen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was prepared from 1 μg of total RNA extracted using iScript reverse transcription supermix for RT-qPCR (Bio-Rad). qPCR was performed using iTaq Universal SYBR green kit (Bio-Rad) and QuantStudio3 Real-Time PCR systems (Thermo Fisher Scientific), and the primers used are listed in Table S2. The fold-change in the expression levels of target genes in treated cells compared to control cells was calculated using the 2-ΔCT method where GAPDH was used as a housekeeping gene.
Myint K., Chuang L.S., Teh Y.X., Mawan N.A., Shi E.J., Mok M.M., Nuttonmanit N., Matsuo J., Li Y., Yang H., Okabe A., Kaneda A., Osato M., So J.B., Yong W.P., Tan P., Yeoh K.G, & Ito Y. (2022). Oncofetal protein IGF2BP1 regulates IQGAP3 expression to maintain stem cell potential in cancer. iScience, 25(10), 105194.
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4

RNA Extraction, cDNA Synthesis, and qPCR Analysis

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Total RNA was extracted using E.Z.N.A. HP Total RNA Kit (Omega Biotek), according to the manufacturer’s instructions and reverse transcribed to cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories). qPCR was performed with iTaq™ Universal SYBR® Green Kit (Bio-Rad Laboratories) on CFX96 RT-PCR Detection Systems. The 18S ribosomal RNA served as the reference gene. The primer sets were purchased from IDT and their sequences are summarized in Supplemental Table S3. The multiplex thermal reaction program consisted of 3 min at 95°C, 40 cycles of 15 s at 95°C, 1 min at 60°C. Target mRNA levels at each time point, relative to the negative control siRNA-treated sample and the internal reference gene, were calculated using the ΔΔCt-method [26 (link)].
Chen L., Bosmajian C, & Woo S. (2024). A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics. Biology Methods & Protocols, 9(1), bpae029.
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5

Real-Time qPCR Analysis of NP Cells

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To further characterise the effects of TAK-242 in mitigating the
response of LPS, the expression of pro-inflammatory, matrix degradation and
ECM-related genes was examined at the end of the 24 h treatment period. Total
RNA was extracted from treated NP cells using the QIAGEN RNeasy kit following
manufacturer’s instructions. Concentration and purity were measured by
NanoDrop, with 260/280 ratios between 1.8 and 2.0 for all samples. 200 ng of
total RNA were converted to cDNA using the iScript cDNA Synthesis kit (BioRad).
Primers listed in Table 2 were designed
using the Integrated DNA Technologies PrimerQuest Tool (Integrated DNA
Technologies). Quantitative PCR was performed using QuantiStudio 6 Flex system
(Applied Biosystems, ThermoFisher Scientific) and iTaq Universal SYBR Green kit
(BioRad) with the following amplification protocol: 95 °C for 30 s
followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Analysis of gene expression was performed by QuantiStudio Real-Time PCR Software
v1.3 software (Applied Biosystems) using the 2−
(ΔΔCt) method to calculate fold change relative to the
untreated group.
Jacobsen T.D., Hernandez P.A, & Chahine N.O. (2021). INHIBITION OF TOLL-LIKE RECEPTOR 4 PROTECTS AGAINST INFLAMMATION-INDUCED MECHANOBIOLOGICAL ALTERATIONS TO INTERVERTEBRAL DISC CELLS. European cells & materials, 41, 576-591.
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6

Quantification of Gene Expression via RT-qPCR

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Total RNA was harvested using TRIzol reagent (Invitrogen) and then isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Total RNA was reverse transcribed with an iScript complementary DNA (cDNA) Synthesis Kit (Bio-Rad). The resulting cDNA was used for real-time PCR using the iTaq Universal SYBR Green Kit (Bio-Rad). β-actin or Gapdh was used as an internal control. Real-time PCR and data collection were performed on a CFX96 instrument (Bio-Rad). Primers for qPCR are listed in Supplementary Table 1.
Yao F., Deng Y., Zhao Y., Mei Y., Zhang Y., Liu X., Martinez C., Su X., Rosato R.R., Teng H., Hang Q., Yap S., Chen D., Wang Y., Chen M.J., Zhang M., Liang H., Xie D., Chen X., Zhu H., Chang J.C., You M.J., Sun Y., Gan B, & Ma L. (2021). A targetable LIFR−NF-κB−LCN2 axis controls liver tumorigenesis and vulnerability to ferroptosis. Nature Communications, 12, 7333.
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7

Total RNA Extraction and qPCR Analysis

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Total RNA was isolated from cells using RNeasy plus micro kit (Qiagen, cat # 74034) according to the manufacturer’s instructions. cDNA was synthesized from RNA using SuperScript III first-strand kit (ThermoFisher, cat # 18080051) according to the manufacturer’s instructions. qPCR was performed on cDNA using iTaq Universal SYBR Green kit (Bio-Rad, cat # 172–5121) and StepOnePlus Real-Time PCR system (ThermoFisher). Primer Sequences are listed in Supplementary Table 1.
Zhu H., Bhatt B., Sivaprakasam S., Cai Y., Liu S., Kodeboyina S.K., Patel N., Savage N.M., Sharma A., Kaufman R.J., Li H, & Singh N. (2019). Ufbp1 promotes plasma cell development and ER expansion by modulating distinct branches of UPR. Nature Communications, 10, 1084.
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8

Quantifying X. euvesicatoria by qPCR

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Real time PCR (qPCR) analyses were performed with iTaq Universal SYBR Green kit (Bio-Rad) according to the manufacturer’s instructions. Xeu 2.4/Xeu 2.5 primers were used [45 (link)]. All amplification reactions were carried out in PikoReal 96 with an initial step at 95 °C for 3 min, followed by 45 cycles of 95 °C for 10 s, 58 °C for 25 s and finally 60 °C for 30 s. To evaluate the number of bacteria by qPCR-based assay, we used a standard curve prepared with a known concentration of X. euvesicatoria 269p suspension.
Shopova E., Brankova L., Ivanov S., Urshev Z., Dimitrova L., Dimitrova M., Hristova P, & Kizheva Y. (2023). Xanthomonas euvesicatoria-Specific Bacteriophage BsXeu269p/3 Reduces the Spread of Bacterial Spot Disease in Pepper Plants. Plants, 12(19), 3348.
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9

Murine IFN-beta ELISA and Mx1 mRNA Quantification

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Murine IFNβ was assayed by ELISA (PBL Verikine). Mx1 mRNA was quantitated in lung tissue (Aurum RNA isolation kit, Bio-Rad) by quantitative PCR (iTaq universal SYBR green kit, Bio-Rad) with Mx1-specific primers (qMmuCID0023356, Bio-Rad), and normalized by parallel amplification of Nidogen-1 (Rotor-Gene, Qiagen).
Tan C.S., Lawler C., May J.S., Belz G.T, & Stevenson P.G. (2016). Type I Interferons Direct Gammaherpesvirus Host Colonization. PLoS Pathogens, 12(5), e1005654.
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10

Real-Time qPCR Analysis of NP Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
To further characterise the effects of TAK-242 in mitigating the
response of LPS, the expression of pro-inflammatory, matrix degradation and
ECM-related genes was examined at the end of the 24 h treatment period. Total
RNA was extracted from treated NP cells using the QIAGEN RNeasy kit following
manufacturer’s instructions. Concentration and purity were measured by
NanoDrop, with 260/280 ratios between 1.8 and 2.0 for all samples. 200 ng of
total RNA were converted to cDNA using the iScript cDNA Synthesis kit (BioRad).
Primers listed in Table 2 were designed
using the Integrated DNA Technologies PrimerQuest Tool (Integrated DNA
Technologies). Quantitative PCR was performed using QuantiStudio 6 Flex system
(Applied Biosystems, ThermoFisher Scientific) and iTaq Universal SYBR Green kit
(BioRad) with the following amplification protocol: 95 °C for 30 s
followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Analysis of gene expression was performed by QuantiStudio Real-Time PCR Software
v1.3 software (Applied Biosystems) using the 2−
(ΔΔCt) method to calculate fold change relative to the
untreated group.
Jacobsen T.D., Hernandez P.A, & Chahine N.O. (2021). INHIBITION OF TOLL-LIKE RECEPTOR 4 PROTECTS AGAINST INFLAMMATION-INDUCED MECHANOBIOLOGICAL ALTERATIONS TO INTERVERTEBRAL DISC CELLS. European cells & materials, 41, 576-591.
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