Express one step superscript qrt pcr universal kit

Manufactured by Thermo Fisher Scientific

The EXPRESS One‐Step Superscript qRT‐PCR Universal Kit is a reagent kit designed for efficient and sensitive real-time reverse transcription-quantitative PCR (RT-qPCR) analysis. It contains all the necessary components for both reverse transcription and quantitative PCR in a single-tube format, simplifying the workflow and minimizing the risk of errors.

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Step One PCR QRT-PCR kit QRT-PCR

6 protocols using express one step superscript qrt pcr universal kit

Quantitative mRNA Analysis in Tissues

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mRNA isolation from flash‐frozen tissues was performed using the RNeasy^® Mini Kit (Qiagen, Venlo, Netherlands). The EXPRESS One‐Step Superscript qRT‐PCR Universal Kit (Invitrogen, Carlsbad, CA) was used for reverse transcription and amplification of SOD1 and MAPT mRNA transcripts. Comparative analysis by the ΔΔC_t method, with GAPDH mRNA transcripts as an internal control, was performed with the ABI PRISM 7500 Fast Real‐Time System (Applied Biosystems, Waltham, MA) (Table S1).

Self W.K., Schoch K.M., Alex J., Barthélemy N., Bollinger J.G., Sato C., Cole T., Kordasiewicz H.B., Swayze E., Bateman R.J, & Miller T.M. (2018). Protein production is an early biomarker for RNA‐targeted therapies. Annals of Clinical and Translational Neurology, 5(12), 1492-1504.

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Viral RNA Extraction and cDNA Synthesis

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The viral RNA used for reverse transcription PCR (RT-PCR) was extracted from serum samples by the PureLink RNA Mini Kit (Ambion, Austin, Texas, USA) following the manufacturer's protocol and quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) with a Qubit RNA BR Assay kit (Invitrogen) following the manufacturer's protocol. Afterwards, viral RNA was used to synthesize cDNA using the EXPRESS One-Step Superscript qRT-PCR Universal kit (Invitrogen) with primers as described elsewhere (Li et al., 2010 (link)).

Diniz D.G., Silva G.O., Naves T.B., Fernandes T.N., Araújo S.C., Diniz J.A., de Farias L.H., Sosthenes M.C., Diniz C.G., Anthony D.C., da Costa Vasconcelos P.F, & Picanço Diniz C.W. (2016). Hierarchical Cluster Analysis of Three-Dimensional Reconstructions of Unbiased Sampled Microglia Shows not Continuous Morphological Changes from Stage 1 to 2 after Multiple Dengue Infections in Callithrix penicillata. Frontiers in Neuroanatomy, 10, 23.

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Quantitative RNA Analysis of Tau Expression

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RNA analyses were performed using quantitative real-time RT-PCR. Total RNA was extracted from brain tissue using a QIAGEN RNeasy Kit (QIAGEN). For total tau analyses, RNA was reverse transcribed and amplified using the EXPRESS One-Step Superscript qRT-PCR Universal Kit (Invitrogen). The qRT-PCRs were run and analyzed on the ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems). Total human and mouse tau expression levels were normalized to mouse GAPDH mRNA levels and analyzed using the ΔΔCt method for relative expression analysis. Primer/Probe sequences: Human Total Tau: Forward 5′-AGA AGC AGG CAT TGG AGA C-3′; Reverse 5′-TCT TCG TTT TAC CAT CAG CC -3′; Probe 5′-/56-FAM/ACG GGA CTG GAA GCG ATG ACA AAA/3IABkFQ/ - 3′, Mouse Total Tau: Forward 5′-GAA CCA CCA AAA TCC GGA GA -3′; Reverse 5′-CTC TTA CTA GCT GAT GGT GAC -3′; Probe 5′-/56-FAM/CCA AGA AGG TGG CAG TGG TCC/3IABkFQ/ - 3′, GAPDH: Forward 5′ – TGC CCC CAT GT TGT GAT G 3′; Reverse 3′ – TGT GGT CAT GAG CCC TTC C – 3′; Probe 5′/56-FAM/ AAT GCA TCC TGC ACC ACC AAC TGC TT /3IABkFQ/ 3′ (IDT).

DeVos S.L., Miller R.L., Schoch K.M., Holmes B.B., Kebodeaux C.S., Wegener A.J., Chen G., Shen T., Tran H., Nichols B., Zanardi T.A., Kordasiewicz H.B., Swayze E.E., Bennett C.F., Diamond M.I, & Miller T.M. (2017). Tau Reduction Prevents Neuronal Loss and Reverses Pathological Tau Deposition and Seeding in Mice with Tauopathy. Science translational medicine, 9(374), eaag0481.

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Quantifying Immune and Proliferative Markers

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Total RNA was purified using RNeasy Plus Mini Kit, according to manufacturer instructions (Qiagen; Valencia, CA), and stored at −80°C. Analysis by qRT- PCR for TLR1, TLR2, TLR4, TLR6, A1, A2A, A2B, A3, Ki-67 and GAPDH mRNA expression was performed using Assays-On-Demand Gene Expression Products and the StepOnePlus real time RT-PCR system (Applied Biosystems; Life Technologies) using Express One-Step Superscript qRT-PCR Universal kit (Invitrogen) and manufacturer's instructions. Briefly, cDNA was synthesized at 50°C for 15 minutes using 25 ng of RNA and subsequently amplified at 95°C for 20 seconds to activate the Platinum^® Taq DNA polymerase enzyme, followed by 40 cycles of 95°C for 15 seconds and 60°C for 20 seconds. The relative expression of the gene of interest was determined using the housekeeping gene GAPDH. In each run, samples were tested in duplicate or triplicate, and repeated as indicated in the Results.

Palani C.D., Ramanathapuram L., Lam-ubol A, & Kurago Z.B. (2017). Toll-like receptor 2 induces adenosine receptor A2a and promotes human squamous carcinoma cell growth via extracellular signal regulated kinases ½. Oncotarget, 9(6), 6814-6829.

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Quantitative RT-PCR Protocols for Cell Culture and Mouse Lung

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For cell culture-based qRT-PCR experiments, total RNA was collected
using Monarch Total RNA Miniprep Kits (NEB). For the mouse lung IAV NP qRT-PCR,
lungs were collected at 4 dpi (with 1200 PFU virus) and homogenized directly in
Trizol, before using chloroform and isopropyl alcohol to complete the RNA
extraction. For the confirmation of siRNA knockdown, measurement of ISG RNA
levels, the comparison of relative MSH6 RNA levels in amiRNA-transfected cells,
and the quantification of mouse lung IAV NP levels, samples were analyzed using
the EXPRESS One-Step Superscript Universal qRT-PCR Kit (Thermo) and
gene-specific TaqMan Expression Assay Probes (Thermo) (Supplementary Table 6). For the MMR
gene panel, the qRT-PCR was performed in two separate steps. First,
single-stranded cDNA was synthesized using the High-Capacity cDNA Reverse
Transcription Kit with RNase Inhibitor (Thermo). Second, cDNA was loaded into
Custom TaqMan Array 96-well Plates (Thermo) we designed to include all major
genes directly involved in the DNA MMR pathway as well as two housekeeping gene
controls for normalization.

Chambers B.S., Heaton B.E., Rausch K., Dumm R.E., Hamilton J.R., Cherry S, & Heaton N.S. (2019). DNA mismatch repair controls the host innate response and cell fate after influenza virus infection. Nature microbiology, 4(11), 1964-1977.

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Quantitative RT-PCR Protocols for Cell Culture and Mouse Lung

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