The AMPLIFYME Probe One-Step No-ROX RT-qPCR Mix is a convenient reaction mixture for fast and reliable quantitative Real-Time PCR using probes, including TaqMan ® , Scorpions ® and molecular beacon probes. It was created for reproducible and efficient first-strand cDNA synthesis and subsequent Real-Time PCR in a single tube. It is the best choice for your probe based Real-Time PCR assays, including singleplex and multiplex gene expression studies.
Additionally, Mu-MLV Reverse Transcriptase and RNase Inhibitor are included in separate tubes.
The use of high-affinity antibody for hot-start polymerase ensures higher specificity, by reducing formation of primer-dimer structures. It allows to obtain wider dynamic range by removing competition for reaction reagents, it also leads to higher sensitivity and reproducibility.
Precisely optimized buffer components ensures optimal conditions for reverse transcriptase and hot-start polymerase activity. Additionally RNase Inhibitor protects RNA from unspecific RNases.
The AMPLIFYME Probe One-Step No-ROX RT-qPCR Mix provides fast, highly specific one-step Real-Time RT-PCR results, giving consistent results across all commonly-used Real-Time PCR platforms.
- gene expression analysis
- genetic profilling
- miRNA profiling / quantification
- mass screening
- RNA viral pathogen detection
- characterization of genetically modified organisms (GMO)
Features & advantages
- Versatile – excellent for various PCR conditions using different Real-Time PCR instruments
- Sensitive – reliable detection of low copies RNA targets
- Reproducible – consistent amplification across a wide dynamic range
- Specific – precisely selected anti-Taq antibody eliminates non-specific amplification
- Efficient – high efficiency in multiplex reactions
- Fast – accurate detection of molecular targets in as fast as 40 minutes (with either two- or three-step cycling profiles)
- Universal – reliable detection of RNA targets from broad range of samples
AMPLIFYME Probe One-Step No-ROX RT-qPCR Mix (AM08) - Manual
AMPLIFYME Probe One-Step No-ROX RT-qPCR Mix (AM08) - Instrument Compatibility
The AMPLIFYME Probe One-Step No-ROX RT-qPCR Mix is compatible with variety of qPCR instrument types that do not require the use of passive reference dye. For ROX-dependent instruments use the AMPLIFYME Probe One-Step Universal RT-qPCR Mix (AM09), which includes additional tubes with High ROX and Low ROX solutions.
Acquisition of high quality, intact RNA, free of genomic DNA and RNase traces, is vital for the synthesis of a full-length cDNA followed by an accurate quantitative analysis (qPCR).
The following recommendations for working with RNA should therefore be followed:
- Maintain aseptic working conditions: use disposable gloves, changing them as frequently as required; use RNase-free consumables; work only in an area assigned for working with RNA and with equipment dedicated for that purpose.
- DNase enzyme may be used if obtaining a DNA-free RNA sample is required.
Special attention should be also paid to PCR products from previous reactions since they represent the greatest danger of contamination. In order to prevent carry-over DNA contamination, it is recommended that the RT-qPCR reaction set-up, PCR amplification and any post-PCR analysis should be carried out in seperate areas with the use of seperate pippets. It is very important that any tubes containing amplified PCR products are not opened in the PCR set-up area.
While analysing similarities between the sensitivity of AMPLIFYME Mixes with competitors’ mixes, it is highly advisable to carry out the amplification process with a 10-fold template dilution series. Loss of signal for low copy targets is the only, distinct survey of sensitivity. Please note that an early Ct value is a determinant of the amplification speed, but not its sensitivity.
RNA contamination with genomic DNA may have an influance on data reliability. Therefore no reverse transcription control should be prepared, by omitting reverse transcriptase in reaction content.
The usage of intron-spanning primers is strongly recommended to avoid amplification of genomic DNA (common DNA contamination from RNA extraction steps).
Store all components at -20°C. Multiple freeze/thawing is not recomended. Aliqoutting can be applied if necessary.
Shipping on dry or blue ice.
The AMPLIFYME Probe One-Step No-ROX RT-qPCR Mix is extensively tested for its performance in different RT-qPCR assays. Free of deoxyribonuclease contamination. Free of DNA contamination.