Product overview Technical/Spec Support/Documents Tips for usage Related products Description The ExcelRT™ One-Step RT-qPCR kit (TaqMan, no ROX) is designed for reverse transcription and quantitative real-time analysis of a specific target RNA by one-step reaction. The ExcelRT™ One-Step RT-qPCR kit (TaqMan, no ROX), consisting of One-Step RT Enzyme Mix and 2X One-Step qPCR Master Mix, is a convenient kit designed for highly efficient cDNA synthesis and highly specific real-time PCR in a single tube. The One-Step RT Enzyme Mix contains a thermostable ExcelRT™ Reverse Transcriptase and a RNAok™ RNase inhibitor. Consequently, One-Step RT Enzyme Mix can reverse transcribe RNA to cDNA at a wide temperature range from 42 to 60°C and active against RNase A, RNase B and RNase C. By containing specialized hot-start Taq DNA polymerase, which greatly reduce primer-dimer formation and can be activated within 2 minutes, the 2X One-Step qPCR Master Mix features high specificity and is suitable for fast cycle program. FeaturesHigh yieldReverse transcription at wide temperature range (42-60°C)High specificity Suitable for fast cycle programWith no ROX reference dyeStorageAliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)Protect from light-20°C for 12 monthsReverse transcription at wide template rangeExcelRT™ One-Step RT-qPCR Kit can quantitative analyze target RNA from a wide range of RNA template input. The amplification plot of one-step RT-qPCR with total RNA templates ranging from 1 pg to 1 ng in quantity, analyzed by using RQ2110 ExcelRT™ One-Step RT-qPCR Kit (TaqMan, ROX) for RT-qPCR amplification.Reverse transcription at wide temperature range (42-60°C)ExcelRT™ One-Step RT-qPCR Kit can quantitative analyze target RNA at a wide temperature range (42-60°C). The amplification plot of one-step RT-qPCR with reverse transcription at temperature range from 42 to 60°C, analyzed by using RQ2110 ExcelRT™ One-Step RT-qPCR Kit for RT-qPCR amplification. The overlapped amplification curves display that ExcelRT™ One-Step RT-qPCR Kit preforms successfully cDNA synthesis at wide temperature range.  Contents ComponentVolume One-Step RT Enzyme Mix400 μl 2X One-Step qPCR Master Mix2 x1 ml StorageAliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)Protect from light-20°for 12 months ManualManual_RQ2200_ExcelRT™ 2X One-Step RT-qPCR Kit (TaqMan, no ROX)SDSSDS_RQ2200 Recommended primer design Amplicon size: 80-150 bp Tm value: around 60°C (calculated with Primer3 software) Primer length: 17-25 mer Sequence: ‒ 45-55% of GC content isrecommended. ‒ Avoid regional high GC or ATcontent ‒ Avoid palindrome sequence ‒ Sequence with G or C at the 3" end is recommended. Specificityof primers should be confirmed through a BLAST search.  Recommended probe design Tm value: 6-10°C higher than primers Probe length: 20-30 mer Sequence: ‒ 35-65% of GC content is recommended. ‒ Avoid regional high GC or AT content ‒ Select the strand contains more C’s than G’s ‒ Avoid palindrome sequence ‒ Avoid a G at the 5" end to prevent quenching of the 5’fluorophore. Specificityof probe should be confirmed through a BLAST search.  Recommended reaction mixture set up for qPCRComponentVolumeFinal concentrationTemplate RNAVaried1 pg – 1 μgForward primer (10 μM)Varied125 – 900 nMReverse primer (10 μM)Varied125 – 900 nMTaqMan Probe (10 μM)Varied125 – 900 nMOne-Step RT Enzyme Mix 2 μl1X2X One-Step Master Mix 10 μl1XddH2Oto 20 μl-Total volume20 µl-*Template amount varies depending on the copy number of target present in the template solution.** The PCR primer and probe concentration for an optimalqPCR reaction may vary according to primers’ and probe’s properties. Recommended qPCRProgramStandard programStepsTemp.TimeCyclesReverse transcription42°C - 60°C (45°C - 55°C is recommended)10min1Enzyme activation 95°C3 min1Denaturation 95°C**15 sec40-50Annealing/ Extension 60°C1 min Fast programStepsTemp.TimeCyclesReverse transcription42°C - 60°C (45°C - 55°C is recommended)5 min1Enzyme activation 95°C20 sec1Denaturation 95°C**3 sec40-50Annealing/ Extension 60°C30 sec  [RP1000] ExcelRT™ Reverse TranscriptaseHigh yieldThermostable, up to 50°C, during first strand synthesisHigh processivity, generating cDNA up to 8 kbReduced RNase H ribonuclease activity[RI1000] RNAok™ RNase InhibitorApplicationRT-PCRcDNA Synthesis in vitro transcription[RP1400] ExcelRT™ Reverse Transcription Kit IIContains all components for reverse transcription High yield Thermostable, up to 50°C Reduced RNase H ribonuclease activity Suitable for real-time PCR[TQ2110] ExcelTaq™ 2X Q-PCR Master Mix (TaqMan, ROX)High sensitivityHigh specificityWith ROX reference dye