qScript Flex cDNA Kit

Flexible first-strand cDNA synthesis system

Features & Benefits

  • Separate components for user-defined RT priming method, (oligo-dT, random hexamer, or GSP)
  • Highly sensitive first-strand cDNA synthesis of RNA sequences ≤ 12kb for quantitative and conventional two-step RT-PCR
  • Broad linear dynamic detection range with limiting (10pg) or plentiful (4ug) total RNA samples
  • Maximize cDNA yield with proprietary Priming Enhancer additive

 

qScript Flex cDNA Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

The qScript Flex cDNA Synthesis Kit is configured in separate components for supporting multiple RT priming strategies. The kit provides optimized, flexible components for priming with oligo-dT(20), random hexamers, gene-specific primer (GSP), or any combination thereof. This unique 5X master mix of buffer, magnesium, stabilizers and dNTPs simplifies reaction assembly and ensures robust and reproducible synthesis of first strand product from 10 pg to 4µg of total RNA or purified polyA+ RNA template. The resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR. Length of cDNA product is dependent upon priming strategy and quality of the RNA template. Oligo dT or GSP can be used for two-step RT-PCR of RNA targets up to 12 kb. Random primer is suitable for RT-PCR of RNA targets less than 1 kb. Any of the three priming methods are suitable for real-time quantitative RT PCR (RT-qPCR).

For one-step RT-PCR of targets less than 5 kb, see our qScript XLT 1-Step RT-PCR Kit

Performance Data

qRT-PCR Dynamic Range


Linearity

qScript Flex cDNA Synthesis Kit with oligo dT primer was used for first-strand cDNA synthesis using log-fold serial dilutions of HeLa cell total RNA from 1 µg to 1 pg. Eight replicate cDNA reactions were performed for each input quantity of RNA. 1/10th of each first-strand reaction was used for qPCR of the ACTB gene using PerfeCTa™ SYBR Green SuperMix.


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Details

  • Contents
    • Master mix, 5X concentrated solution of optimized reaction buffer, magnesium, dNTP blend, proprietary stabilizers and additives
    • Oligo dT(20), 10X concentrated with Priming Enhancer
    • Random Primer, 10X concentrated with Priming Enhancer
    • Priming Enhancer, 10X concentrated
    • Stabilized qScript Reverse Transcriptase with ribonuclease inhibitor protein (RIP)
    • Nuclease-free water
  • Storage & Handling
    Store components in a constant temperature freezer at -25°C to -15°C upon receipt. After thawing, mix thoroughly before using. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
    Thaw completely on ice then pulse vortex to mix and briefly spin-down to collect contents before opening tube. Concentrated reagents are viscous. When drawing out material, touch pipette tip to liquid interface but do not immerse.
  • Related Resources
    Product Manuals
    Technical Notes
    FAQs
    What is the Flex Kit? How is it different from the cDNA synthesis Kit? Can we use either an oligo dT or a random priming method?
    The Flex Kit supports the use of multiple priming strategies. When using a combination of the oligo dT primer solution and random primer solution, the linear dynamic range and qRT-PCR performance is comparable to that of the qScript cDNA Synthesis Kit. In addition to retaining high performance with limiting amounts of RNA, the Flex kit was also optimized to provide long cDNA synthesis with oligo dT primer. So its composition is different from that of the cDNA synthesis Kit. When using oligo dT primer alone, it is unquestionably one of the easiest to use and most sensitive cDNA kits on the market
    What is the qScript™ Flex cDNA Kit? How is it different from the qScript™ cDNA Synthesis Kit?
    The qScript Flex cDNA Synthesis Kit is an easy-to-use and highly efficient kit for the synthesis of first-strand cDNA that enables your choice of cDNA priming method. Various RT-PCR applications may require different priming strategies for optimal performance and this kit provides optimized reagents for priming with oligo(dT)20, random primer, gene-specific primer (GSP), or any combination thereof. Therefore, qScript™ Flex cDNA Kit is ideal for developing the best protocol for your application. The qScript™ Flex cDNA Kit also allows the use of Oligo dT or gene-specific primers (GSP) for long RT-PCR of RNA targets up to 12 kb. The qScript cDNA Synthesis Kit consists of an optimized blend of random and oligo(dT) primers. It provides robust, consistent and unbiased first-strand synthesis over a broad range of RNA template concentrations. qScript™ cDNA Synthesis Kit is well suited for short RT-PCR apllications (i.e. qRT-PCR) and is not recommended for amplification of RNA's longer than 1kb. When using a mixture of the Oligo dT and Random Primer solutions, the qScript™ Flex cDNA Kit achieves first-strand synthesis over a broad range of RNA template concentrations and its performance is comparable to that of the qScript™ cDNA Synthesis Kit. With both kits, the resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR.
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    Publications
    A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex
    Margaret L. Rodgers, Oxford Journals - 2016
    Abstract
    The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.
    Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma
    A. Bergeron, Andrology - 2016
    Abstract
    The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5′AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/μg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2 ± 14.8 fmoles/min/μg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.
    microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure
    Gesche K.Gerresheim, Cellular and Molecular Life Sciences - 2016
    Abstract
    We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3′ untranslated region (3′UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3′UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3′UTR target site inhibits translation directed by the HCV 5′UTR. Thus, the miR-122 target sites in the 3′-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.
    An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery
    Aline Koch, PLOS Pathog - 2016
    Abstract
    Author Summary RNA interference has emerged as a powerful genetic tool for scientific research. The demonstration that agricultural pests, such as insects and nematodes, are killed by exogenously supplied RNA targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNA signals. We show that spraying barley with a 791 nt long dsRNA ( CYP3 -dsRNA) targeting the three fungal ergosterol biosynthesis genes ( CYP51A , CYP51B , CYP51C ), whose respective proteins also are known as azole fungicide targets, efficiently inhibited the necrotrophic fungus Fusarium graminearum in directly sprayed and systemic leaf tissue. Strong inhibition of fungal growth required an operational fungal RNA interference mechanism as demonstrated by the fact that a Fusarium DICER-LIKE-1 mutant was insensitive to CYP3 -dsRNA in systemic, non-sprayed leaf areas. Our findings will help in the efficient design of RNAi-based plant disease control. We provide essential information on a fundamentally new plant protection strategy, thereby opening novel avenues for improving crop yields in an environmentally friendly and sustainable manner.
    Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis
    Rémi Labrecque, Molecular Reproduction and Development - 2016
    Abstract
    Follicle size is recognized as a predictor of the potential for the enclosed oocyte to yield an embryo following in vitro maturation and in vitro fertilization. Oocytes from larger follicles are more likely to reach the blastocyst stage than those from smaller follicles. A growing oocyte accumulates all the transcripts needed to ensure development until the maternal embryonic transition, and this accumulation must be completed before the period of transcriptional arrest. Accordingly, the transcriptomes of bovine germinal-vesicle-stage oocytes collected from follicles of increasing sizes (<3, 3–5, >5–8, and >8 mm) were evaluated, using the EmbryoGENE bovine transcriptomic platform (custom Agilent 4 × 44 K), to better understand transcriptional modulation in the oocyte as the follicle becomes larger. Microarray analyses revealed very few differences between oocytes from small follicles (<3 vs. 3–5 mm), whereas an important number of differences were detected at the mRNA level between oocytes from larger follicles. Weighted gene correlation network analysis allowed for the identification of several hub genes involved in crucial functions such as transcriptional regulation (TAF2), chromatin remodeling (PPP1CB), energy production (SLC25A31), as well as transport of key molecules within the cell (NAGPA, CYHR1, and SLC3A12). The results presented here thus reinforce the hypothesis that developmental competence acquisition cannot be seen as a simple one-step process, especially in regards to the modulation of mRNA. Mol. Reprod. Dev. 83: 558–569, 2016. © 2016 Wiley Periodicals, Inc.
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