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QuantiFast Probe Assays

For qPCR and qRT-PCR gene expression analysis using predesigned, bioinformatically-verified assays together with QuantiFast Kits

  • Guaranteed results with genomewide, predesigned assays
  • Efficient, reliable detection of targets less than 100 bp in size
  • Highly suited for detection of RNA from FFPE samples
  • Sensitive detection of target and reference gene in one tube
  • Suitable for both two-step and one-step RT-PCR

QuantiFast Probe Assays are predesigned assays based on dual-labeled hydrolysis probe detection for gene expression analysis. Each assay for a specific gene is supplied as a lyophilized mix of forward and reverse primers and probe that can easily be reconstituted to obtain a 20x assay solution. Used in combination with QuantiFast Kits, QuantiFast Probe Assays enable rapid real-time RT-PCR quantification of cDNA or RNA targets in a singleplex format (FAM reporter dye) or duplex format (FAM and MAX reporter dyes). QuantiFast Probe Assays are available for every human, mouse, or rat gene. They are designed to enable amplification and detection of all targets, including ones that are less than 100 bp in size, which makes them highly suited for use with degraded starting material such as formalin-fixed, paraffin-embedded (FFPE) analytes. For convenience, the master mix can be stored at 2–8°C.

Search in GeneGlobe
You can search for the following terms:
  • Entrez Gene IDs (e.g., 835)
  • RefSeq IDs (e.g., NM_032983, NP_116765)
  • Gene symbols (e.g., CASP2)
  • Cat. no. (e.g., SI00299551, QT01342509)
  • Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)
  • CpG loci identification numbers (CG#) (e.g., CG17753661)

Do not enter species information in the Search box. Use the drop-down list to select your species of interest.
When searching for miRNAs, do not omit the hyphens. Use hyphens or spaces (e.g., search for hsa-let-7b or hsa let 7b. Do not search for hsalet7b).

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Reference assays for dual-labeled probe detection - FAM labeled

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Mouse
Rat
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QuantiFast Probe Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Highly efficient real-time RT-PCR detection using RNA from an FFPE sample.
Total RNA (1 ng, 100 pg, 10 pg, and 1 pg) was purified from a breast tissue FFPE sample using the RNeasy FFPE Kit. Transcripts of the human MUC1 gene were amplified and detected on the Rotor-Gene Q cycler using the QuantiFast Probe RT-PCR Plus Kit and the QuantiFast Probe Assay (blue curves) or a predesigned assay from Supplier AII (red curves). Results from the QuantiFast Assay showed lower CT values and higher sensitivity compared with results using the assay from Supplier AII. Template amounts as low as 1 pg were detectable using the QuantiFast Probe Assay (1 pg was undetectable using the assay from Supplier AII).
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Highly efficient real-time RT-PCR detection of degraded RNA.
Total RNA (10 ng to 100 pg) from HeLa cells with a RIN value of 4.7 was amplified and detected in one-step RT-PCR on the ABI 7500 Fast cycler using [A] a QuantiFast Probe Assay for the human CENPM gene in combination with the QuantiFast Probe RT-PCR Plus Kit (amplicon size: 76 bp) or [B] a predesigned assay and kit from Supplier AII (amplicon size: 111 bp). Results from QuantiFast Reagents showed lower CT values and higher sensitivity compared to results using reagents from Supplier AII.
Performance
QuantiFast Probe Assays are designed for gene expression analysis and, in addition, are highly suited for FFPE analytes (see figure "Highly efficient real-time RT-PCR detection using RNA from an FFPE sample" and "Highly efficient real-time RT-PCR detection of degraded RNA").
Principle

QuantiFast Probe Assays are predesigned, genomewide assays for human, mouse, or rat genes. They are designed using a proprietary algorithm to enable amplification and detection of any RNA and cDNA targets. In addition, they can detect fragments of less than 100 bp in size with high efficiency and reliability. Each QuantiFast Probe Assay consists of a premixed primer pair and a dual-labeled probe. Two dye labels are available: FAM (for all genes) and MAX (for selected reference genes). QuantiFast Probe Assays can be easily ordered at the GeneGlobe Web portal.

For single or duplex one-step RT-PCR, QuantiFast Probe Assays are used in combination with the QuantiFast Probe RT-PCR Plus Kit for the detection of RNA. After addition of the master mix, reverse transcription and real-time PCR take place in the same tube. This kit offers an integrated genomic DNA removal step that effectively eliminates genomic DNA contamination to avoid false-positive signals. Removal of genomic DNA is particularly important in samples containing highly degraded RNA (e.g., RNA from FFPE tissue samples), where the presence of genomic DNA frequently results in false positive RT-PCR signals.

For single or duplex two-step RT-PCR, QuantiFast Probe Assays are delivered in combination with the QuantiFast Probe PCR Kit or the QuantiFast Multiplex PCR Kit, respectively. QuantiFast Probe Assays, used together with QuantiFast Kits, provide reliable and highly sensitive detection of cDNA targets and eliminate tedious design and optimization steps.

For cDNA synthesis, the QuantiTect Reverse Transcription Kit is recommended. The QuantiTect Reverse Transcription Kit provides an easy and fast cDNA synthesis procedure and includes a genomic DNA elimination step to avoid false-positive signals.

Procedure

Each QuantiFast Probe Assay is supplied as a lyophilized mix of forward and reverse primers and a dual-labeled probe. QuantiFast Probe Assays should be reconstituted in TE, pH 8.0 (provided) to give a 20x mix, which is then added to the master mix supplied with a QuantiFast Kit. The reaction mix is aliquoted into PCR tubes, wells, or Rotor-Discs, and the individual template samples are then added.

For guaranteed results in gene expression analysis, QuantiFast Probe Assays are provided with the following kits shown in the table.

Two-step and one-step qRT-PCR kits for use with QuantiFast Probe Assays
Two-step qRT-PCROne-step qRT-PCR
Singleplex QuantiFast Probe PCR Kit QuantiFast Probe RT-PCR Plus Kit
Duplex QuantiFast Multiplex PCR Kit QuantiFast Probe RT-PCR Plus Kit

For both singleplex and duplex two-step qRT-PCR assays, cDNA can be prepared using the QuantiTect Reverse Transcription Kit, which provides cDNA synthesis with integrated removal of genomic DNA contamination.

Naming

Each QuantiFast Probe Assay available for a particular gene is given a number that appears in the product name after the gene symbol. For more information, please see the GeneGlobe FAQs.

Applications

QuantiFast Probe Assays are highly suited for applications such as gene expression analysis of targets less than 100 bp in size, which makes them highly suited for use with degraded starting material, including FFPE analytes.

For guaranteed results in gene expression analysis, QuantiFast Probe Assays are provided with the following kits:

  • QuantiFast Probe RT-PCR Plus Kit: one-step RT-PCR
  • QuantiFast Probe PCR Kit: two-step RT-PCR, singleplex
  • QuantiFast Multiplex PCR Kit: two-step RT-PCR, duplex
Features
Specifications
Applications Gene expresssion analysis: Real-time quantification of RNA or cDNA targets
Detection Sequence-specific probes: dual-labeled probes
Format Lyophilized primer-probe pre-designed assay
Reaction type Real-time one-step RT-PCR including a genomic DNA removal step or real-time two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA or cDNA
Single or multiplex Single or duplex
Species Human, mouse and rat
Thermal cycler All real-time cylcers compatible with duplex RT-PCR, e.g. Rotor-Gene Q or cyclers from Agilent, Applied Biosystems, BioRad, Roche
With or without ROX Master Mix is provided without ROX dye, 2 separate ROX solutions are included to be compatible with any real-time cycler
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