Lyo-Ready™ dUTP 1-Step RT-qPCR Master Mix
Meridians Lyo-Ready™ dUTP 1-Step RT-qPCR Mix is a glycerol-free mix, ideal for developing ambient temperature, lyophilized, multiplex qPCR and RT-qPCR molecular diagnostic tests and are suited for high-throughput, automated platforms.
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High efficiency and sensitivity from both DNA (red) and RNA (blue) templates
Amplification of both Rotavirus A (dsRNA) and Adenovirus (dsDNA) in a single multiplexed RT-qPCR assay, from inactivated crude viral lysates. The result illustrates that both the DNA and RNA viruses were amplified with high efficiency and sensitivity, demonstrating the ability of Lyo-Ready™ 1-Step RT-qPCR Virus Mix to detect low-copy number RNA and DNA targets simultaneously from a single sample.
Molecular diagnostic tests are progressively moving towards lyophilized formats. There are several advantages for this, including ambient temperature shipping and storage, extended shelf-life, reduced operating steps and potential errors and increased flexibility in sample volume. In order to be compatible with drying, however, enzyme preparations must be glycerol-free and include specialized excipients that preserve the mixture as it is exposed to various conditions including freezing, vacuum, and dehydration.
Lyo-Ready™ dUTP 1-Step RT-qPCR Mix is a 2x RT-qPCR master mixes and can be used for the development of RNA based molecular diagnostic tests, it contains a separate lyo-compatible MMLV-RT and enzyme dilution buffer. The dTTP in the dNTPs has been substituted for dUTP, so that Uracil DNA Glycosylase (UDGase) (MDX054) can be added to remove background PCR product contamination. This makes Lyo-Ready™ dUTP 1-Step RT-qPCR Mix ideal for developing highly sensitive RT-qPCR diagnostic tests, such as for detecting RNA viruses at very low levels.
|Description||Glycerol-free master mix containing Taq polymerase, RNase Inhibitor, reaction buffer, dNTP/dUTP mix, MgCl2 and lyo-excipients and separate reverse transcriptase and dilution buffer, developed to remove background PCR product contamination.|
|Format||Clear, colorless solution|
|Hot Start||Antibody mediated|
|Application||High throughput, qPCR, two-step RT-qPCR|
|Sample type||cDNA, DNA|
|Mix stability||See outer label|
|Assay stability||Up to 24 months at ambient temperature following lyophilization|
|Consistency||±0.5 Ct variance between test and reference sample|
|DNA Contamination||None detected in PCR amplification with traces overlay with the negative control on E. coli and mouse genomic DNA specific targets.|
|DNase Contamination||No detectable degradation|
FAQs: Lyo-Ready qPCR and RT-qPCR Master Mixes
In one-step RT-qPCR or reverse transcription qPCR, cDNA synthesis and qPCR are performed in a single reaction tube. In two-step qPCR, cDNA is synthesized in one reaction tube, and aliquots of the cDNA is then used for a subsequent qPCR experiment.
• Accurate representation of target copy number
• Simple and rapid
• Fewer pipetting steps (reducing possible errors and contamination)
• Best option for high-throughput screening
• Best method when only a few assays are run repeatedly
• Multiplex qPCR of gene of interest and control can be done in single well, from same RNA sample
There are several other advantages of one-step reactions, these include limited sample handling and reduced bench time, which helps to decrease chances for pipetting errors and cross contamination between reverse transcription and qPCR steps. This method is quick to set up and makes processing multiple RNA samples easy (especially when using liquid handling robotics), when you are amplifying only a few genes of interest. It is therefore ideal for high throughput screening laboratories where only a few reverse transcription qPCR assays are run repeatedly, using well-established reaction conditions, with the added advantage that multiplex qPCR of the gene of interest and control genes can be done in single well, from same RNA sample.
Gene-specific primers are required for generating the cDNA and for subsequent amplification in one tube, however this reduces experimental variation, since both enzymatic reactions take place under the same conditions, making one-step qPCR highly reproducible.
One-step RT-PCR requires careful evaluation to prevent primer dimer formation, because the primers will be present during the lower temperature conditions of the RT reaction as well as the qPCR cycling. This is particularly important in multiplex qPCR assays.
As qPCR can amplify tiny amounts of DNA, preventing contamination is essential, as even small amounts of qPCR contamination can produce false positives. qPCR contamination can include cross-contamination from other samples, contamination from elsewhere in the laboratory, and carryover contamination from amplification products and primers used in prior qPCR experiments. The latter causes most of the false positive results seen in qPCR. Preventing contamination can be difficult and so laboratory procedures must be in place to ensure DNA from one assay is not re-amplified in subsequent assay.
Uracil DNA Glycosylase can specifically degrade products that have already been through the qPCR process and have incorporated dUTP, leaving native nucleic acid templates intended for amplification intact. Uracil DNA Glycosylase activation occurs as the first step of qPCR at a 50°C incubation for 2 minutes and then the Uracil DNA Glycosylase is denatured when the temperature increases to 95°C during hot-start activation.
Uracil-DNA glycosylase (UDG or UDGase) is a family of enzymes comprising six sub-families. Family I UDGase enzymes are called UNG (uracil-N-glycosylase gene), so there is no difference, the terms UDG and UNG are commonly used interchangeably because they perform the same function in qPCR—namely to prevent carryover qPCR contamination.
No-RT control reactions are useful for determining issues that may arise from the amplification of genomic DNA that may be present in a sample. However, many RNA transcripts are present at low abundance and for these transcripts, it is recommended to perform a No-RT control reaction if primer sets do not span exon-exon junctions. If the primers do span exon-exon junctions, or genomic DNA is not likely to interfere (when looking at RNA viruses in a sample for example) a No-RT control is not required.
Following lyophilization in the presence or absence of primers and probes, the Lyo‑Ready™ 1-Step RT-qPCR Virus Mix is stable for a minimum of 24 months at ambient temperature (17 – 23 °C). Following rehydration, the assay reproducibility, sensitivity and robustness will be the same as for a freshly made liquid mix, making the mixes ideal for point-of-care molecular diagnostic tests and microfluidic qPCR devices.
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