Luna universal one step rt qpcr kit

Manufactured by New England Biolabs

Sourced in United States, Germany, United Kingdom, Italy

The Luna Universal One-Step RT-qPCR Kit is a reagent system designed for reverse transcription and real-time quantitative PCR (RT-qPCR) analysis. It enables the conversion of RNA to cDNA and subsequent amplification and detection of target sequences in a single reaction.

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Luna Universal Probe One-Step RT-qPCR Kit (122 citations) NEB Luna Universal Probe One-Step RT-qPCR Kit (12 citations) Luna Universal qPCR kit (8 citations) Luna® Universal One-Step RT-qPCR (5 citations) Luna one-step RT-qPCR kit (5 citations) Luna Universal One-Step qPCR Kit (4 citations) Sybr green Luna Universal one-step RT-qPCR kit (3 citations) Luna Universal One-Step RT qPCR Master Mix kit (2 citations) Luna Universal Probe one-step reverse transcription-quantitative PCR (RT-qPCR) kit (2 citations)

270 protocols using luna universal one step rt qpcr kit

mCherry-irg1l Fusion Protein Analysis

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qRT-PCR for RNA-seq verification was performed using 10 ng of total RNA in a one-step qRT-PCR [Luna Universal One-Step RT-qPCR Kit, New England Biolabs (NEB)] and performed on a Roche LightCycler 96 system. Primers were designed for verifying some of the up-regulated and immune-related genes (see table S1 for primers used). All graphs are representative of two independent experiments with three technical replicates.
Functional testing of mCherry-irg1l fusion protein was performed by RNA overexpression, followed by qRT-PCR assays of mmp9 target gene induction (22 (link)). Briefly, untagged irg1l and mCherry-irg1l were subcloned into pCS2⁺ vectors and used as templates for in vitro transcription. Equimolar amounts of each RNA were injected into one-cell–staged embryos, and total RNA was extracted from 50 embryos at 28 hpf using TRIzol reagent (catalog no. 15596026, Invitrogen) following the manufacturer’s recommendations. Total RNA (100 ng) was used in a one-step qRT-PCR (Luna Universal One-Step RT-qPCR Kit, NEB) and performed on a Roche LightCycler 96 system. All analyses were carried out in triplicate. Results are representative of two independent experiments with three technical replicates each. See table S1 for primer sequences.

Van Gennip J.L., Boswell C.W, & Ciruna B. (2018). Neuroinflammatory signals drive spinal curve formation in zebrafish models of idiopathic scoliosis. Science Advances, 4(12), eaav1781.

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Quantifying PEDV Viral RNA Copy Number

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Total RNA was isolated from VeroE6-APN cells transfected with siRNA by using RNeasy kit (Qiagen) and then subjected to RT-qPCR using Luna^® Universal One-Step RT-qPCR Kit (NEB, USA) according to the manufacturer’s protocol. RT-qPCR was performed using the CFX96 Touch™ Real-Time PCR Detection System (BioRad). The housekeeping gene GAPDH was used as an internal control. Normalized data from each sample relative to zero were compared by the threshold cycle (∆∆CT) method [16 (link)]. All primers used are listed in Table 2.
To determine the viral RNA copy number, the supernatant containing virus collected from infected cells at indicated time points was subjected for viral RNA isolation using a Luna^® Universal One-Step RT-qPCR Kit (NEB) following the manufacturer’s instructions. The primers specific for PEDV M gene and quantification of the virus copy number were described previously [7 (link)].

Kaewborisuth C., Yingchutrakul Y., Roytrakul S, & Jongkaewwattana A. (2019). Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Interactome Reveals Inhibition of Virus Replication by Cellular VPS36 Protein. Viruses, 11(4), 382.

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Validating miRNA Expression in AMI

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To validate the reliability of RNA sequencing data, differentially expressed miRNAs were randomly selected and qRT‐PCR was performed to examine the expression level of miRNAs. Total RNA was extracted from the plasma using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Extracted RNA was converted to cDNA, and qRT‐PCR reactions were performed using Luna Universal One‐Step RT‐qPCR kits (New England Biolabs). We verified the expression of these miRNAs from PBMCs of AMI patients and healthy controls with qRT‐PCR using glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as the reference gene with the 2^−ΔΔCT method.

Zhong Z., Wu H., Zhong W., Zhang Q, & Yu Z. (2019). Expression profiling and bioinformatics analysis of circulating microRNAs in patients with acute myocardial infarction. Journal of Clinical Laboratory Analysis, 34(3), e23099.

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Validating lncRNA Expression via qRT-PCR

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To validate the reliability of RNA sequencing data, differentially expressed lncRNAs were randomly selected and qRT-PCR was employed to examine the expression level of lncRNAs. Total RNA were extracted from the PBMCs using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. And qRT-PCR reactions were performed by using Luna Universal One-Step RT-qPCR kits (New England Biolabs, MA). The PCR reactions were carried out by the conditions: 15 s at 55 °C and 1 minute at 95 °C, followed with 40 cycles for 10 seconds at 95 °C and 30 seconds at 60 °C, and 30 seconds at 50 °C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for measurement of lncRNAs and the relative expression levels of candidate lncRNAs were calculated using the 2^−ΔΔCT equation. At least triple experiments were subjected to qRT-PCR verification.

Zhao P., Wu H., Zhong Z., Zhang Q., Zhong W., Li B., Li C., Liu Z, & Yang M. (2018). Expression profiles of long noncoding RNAs and mRNAs in peripheral blood mononuclear cells of patients with acute myocardial infarction. Medicine, 97(41), e12604.

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Quantifying PBRM1 Expression via RT-qPCR

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At 24, 48, 72, 96, 120, 144, and 168 h after transfection, RNA was isolated from pelleted cells using Qiagen RNEasy Mini Kits (Hilden, GER). RT-qPCR was performed using Luna Universal OneStep RT-qPCR kits (New England Biolabs, Ipswich, USA) on a Quantstudio 3 (Thermo Fischer, Waltham, USA) according to the manufacturer’s instructions. RT-qPCR primers for PBRM1 and GAPDH were obtained from Eurofins Genomics (Ebersberg, Germany; Sequence GAPDH-primer (Forward: 5′-CAA-TGA-CCC-CTT-CAT-TGA-CC-3′, and Reverse: 5′-TTG-ATT-TTG-GAG-GGA-TCT-GG-3′) and PBRM1-primer (Forward: 5′-AGG-AGG-AGA-CTT-TTC-AAT-CTT-CC-3′, and Reverse: 5′-CTT-CGC-TTT-GGT-GCC-CTA-ATG-3′). Relative gene expression was calculated by the ΔΔCT method^{44 (link),45 (link)}, using GAPDH expression levels to normalize PBRM1 expression levels.

Zimmer K., Kocher F., Untergasser G., Kircher B., Amann A., Baca Y., Xiu J., Korn W.M., Berger M.D., Lenz H.J., Puccini A., Fontana E., Shields A.F., Marshall J.L., Hall M., El-Deiry W.S., Hsiehchen D., Macarulla T., Tabernero J., Pichler R., Khushman M., Manne U., Lou E., Wolf D., Sokolova V., Schnaiter S., Zeimet A.G., Gulhati P., Widmann G, & Seeber A. (2023). PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system. NPJ Precision Oncology, 7, 64.

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Quantifying lncRNA Expression in STEMI

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cDNA was synthesized and Real-Time Quantitative PCR (RT-qPCR) of lncRNAs expression level was performed using Luna Universal One-Step RT-qPCR kits (New England Biolabs, MA) according to the manufacturer's protocol. The PCR were performed on the LightCycler 480/II machine (Roche, Mannheim, Germany) and the PCR conditions were set as follow: 1 cycle of 10 minutes at 55 °C, 1 cycle of 1 minute at 95 °C, 45 cycles of 10 second at 95 °C and 30 seconds at 60 °C, and 1 cycle of 1 minute at 60 °C finally. Gene expression levels were quantified were calculated using the method of 2^−ΔΔct and normalized to the internal control of the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).^{[25 (link)]} The level of each lncRNAs in STEMI patients was expressed as the fold changes against the averaged level of the same lncRNA in NSTEMI patients.

Zhong Z., Hou J., Zhang Q., Li B., Li C., Liu Z., Yang M., Zhong W, & Zhao P. (2018). Differential expression of circulating long non-coding RNAs in patients with acute myocardial infarction. Medicine, 97(51), e13066.

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Ovary RNA Extraction and RT-qPCR

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Five one to three-day old females were incubated with males in yeasted vials overnight. Five pairs of ovaries were dissected from two to four-day old females in ice-cold 1X PBS, and flash-frozen in liquid nitrogen. Total RNA was extracted using the Monarch Total RNA Miniprep Kit (NEB), according to the manufacturer’s instructions for RNA purification from “Tissues”. RT-qPCR was performed on 10 ng total RNA using the Luna Universal One-Step RT-qPCR Kit (NEB, E3005L). bin3, 7SK, U6, and rp49 mRNAs were reverse transcribed and amplified with primers UP0493 + UP0118, UP0146 + UP0147, OW1123 + OW1124, and UP0113 + UP0114, respectively (see Table S4 for primer sequences). Reactions were performed using a CFX Opus 384 Real-Time PCR System (Bio-Rad).

Palumbo R.J, & Hanes S.D. (2023). Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster. bioRxiv.

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Quantification of HCV RNA and Genes

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Total RNA extraction was performed with the RNeasy mini Kit (Qiagen). Quantification of HCV genome RNA and FADS1, FADS2, GPX4, AIFM2, ACSL4, ACTB and ALOX genes was carried out by a One-step quantitative RT-PCR analysis with the Luna Universal One-Step RT-qPCR Kit (NEB) using the primer pairs listed in Table S1.

Yamane D., Hayashi Y., Matsumoto M., Nakanishi H., Imagawa H., Kohara M., Lemon S.M, & Ichi I. (2021). FADS2-dependent fatty acid desaturation dictates cellular sensitivity to ferroptosis and permissiveness for hepatitis C virus replication. Cell chemical biology, 29(5), 799-810.e4.

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Quantifying Staphylococcus aureus RNA Levels

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The eluted bacterial total RNA was qualified with a 2100 Bioanalyzer (Agilent) using an RNA 6000 Pico chip and analyzed by quantitative reverse transcription PCR (RT-qPCR) using the CFX Connect Real-Time PCR system (Bio-Rad) as follows. To convert RNA to cDNA and then quantify the cDNA in a single step, a Luna® Universal One-Step RT-qPCR Kit (New England BioLabs) was used with a FAM dye-labeled probe for ribonuclease P RNA (rnpB) in S. aureus (Taqman® gene expression assays, ID number: Ba04646259_s1, Thermo Fisher Scientific) following the manufacturer's protocol. RT-qPCR was performed with 1 cycle of 95 °C for 10 min and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Quantification of gene expression was based on the cycle quantification (Cq) value compared with that of the positive controls extracted by the RNeasy Mini Kit from Qiagen. The delta Cq was calculated as (Cq of captured sample on chip) - (Cq of control sample) using CFX Maestro 1.1 software (Bio-Rad) and Microsoft Excel (Microsoft Corp., USA). The relative quantity of rnpB gene expression was determined by a delta-delta Cq calculation as 2^(-(treated sample delta Ct-control sample delta Ct)) 29 (link).

Park J., Woo S., Kim J., Lee H., Yoo Y.E, & Hong S. (2021). Rapid and simple single-chamber nucleic acid detection system prepared through nature-inspired surface engineering. Theranostics, 11(14), 6735-6745.

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Fetal Testis RNA Extraction and RT-qPCR

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Fetal testes were dissected, isolated from mesonephros, snap frozen in liquid nitrogen and stored at −80°C until use. Whole-testis total RNA was extracted using RNeasy Mini Kit (Qiagen, Les Ulis, France). RT-qPCR was performed on 5 ng RNA aliquots using Luna Universal One-Step RT-qPCR Kit (New England Biolabs, Evry, France), according to the manufacturer's instructions. The primers used are listed in Table S8. Triplicates of at least four samples were used for each genotype at each stage. The relative transcript levels were determined using the ΔΔCt method, and normalized to Acta, the expression of which is not affected by ablation of Nr5a1.

Souali-Crespo S., Condrea D., Vernet N., Féret B., Klopfenstein M., Grandgirard E., Alunni V., Cerciat M., Jung M., Mayere C., Nef S., Mark M., Chalmel F, & Ghyselinck N.B. (2023). Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis. Development (Cambridge, England), 150(24), dev201710.

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