In a one-step qRT-PCR reaction, reverse transcription and quantitative PCR are performed as a single, closed tube assay, reducing the risk of contamination. Fewer pipetting steps and ease-of-use make one-step qRT-PCR ideal for routine, high-throughput screening. In two-step qRT-PCR reactions, reverse transcription and quantitative PCR are performed as separate reactions in separate tubes. Two-step qRT-PCR provides more flexibility since cDNA can be archived and used for additional real-time PCR reactions, eliminating the need to continually isolate RNA. However this flexibility comes with an increased risk of contamination and prolonged set up time.
KAPA SYBR DNA Polymerase is an engineered variant of Taq DNA polymerase. It was specifically engineered for use in SYBR Green- based qPCR through a process of molecular evolution. KAPA SYBR DNA Polymerase is less inhibited by SYBR Green I dye and is more processive than wild-type Taq DNA Polymerase, resulting in increased sensitivity, fluorescence and speed. The enzyme has an integrated antibody-mediated hot start to remain inactive during reaction set-up, minimizing the formation of non-specific products.
The 50X KAPA RT Mix contains wild-type M-MuLV Reverse Transcriptase that allows cDNA synthesis to occur at low temperatures. In addition, an RNase inhibitor is included in this enzyme mix to prevent RNA degradation due to RNase contamination.
KAPA SYBR FAST One-Step qRT-PCR Kits can be used with most RNA as template, (e.g., human, mouse, rat and bacteria.)
Suggested input quantities for KAPA SYBR FAST One-Step qRT-PCR assays:
All isolated RNA can be assessed electrophoretically on a denaturing 2% agarose gel containing ethidum bromide. The gel should contain sharp 28S and 18S rRNA bands with no smearing at their low molecular weight edge. If utilizing the Agilent BioAnalyzer, the RNA Integrity Number (RIN) should be above 5.0.
For certain real-time cyclers, the presence of ROX reference dye in real-time PCR compensates for non-PCR-related variations in fluorescence detection. Fluorescence from ROX reference dye does not change during the course of real-time PCR, but provides a stable baseline to which PCR-related fluorescent signals are normalized. Thus, ROX dye compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or to differences in well position. The use of ROX dye is necessary for all instruments from Applied Biosystems and is optional for the Mx3000P, Mx3005P, and Mx4000®. Instruments from Bio-Rad/MJ Research, Cepheid, Corbett Research, Eppendorf, and Roche do not require ROX dye. The presence of ROX dye in the master mix does not interfere with real-time PCR on any instrument, since the dye is not involved in the reaction and has an emission spectrum different from that of SYBR Green I.
dUTPs are supplied in a separate tube to offer optional treatment with Uracil-DNA glycosylase (UDG) for the prevention of carry-over contamination in subsequent reactions. UDG is not supplied in KAPA SYBR FAST qRT-PCR Kits.
It is not recommended to use UDG when performing reverse transcription. When using a dNTP mix with dUTP in a reverse transcription reaction, uracil will be incorporated into the cDNA generated from the RNA template. The UDG will cleave the DNA containing dUTP residues and result in inefficient or no amplification.
No. The reactions must be set up on ice. The 50X KAPA RT Mix is not thermostable; hence all reaction components must be kept cold during set up.
Optimal cDNA synthesis using wild-type M-MuLV RT occurs at 42ºC, however in cases of difficult templates, temperature adjustment between 42º–50ºC can be performed.
Only gene-specific primers can be used in a One-Step qRT-PCR system.
Yes, any residual DNase activity has the potential to degrade cDNA product and thereby reduce the amount of template available for qPCR amplification. DNase will not be active during PCR cycling, however, it could degrade primers prior to the cycling process
If the PCR efficiency is still sub-optimal after adjusting combined annealing/extension times and temperatures or when annealing temperatures of primers are less than 60ºC, it is advisable to convert to a 3-step PCR protocol. If the optimal annealing temperature is 55ºC, then perform annealing for 20 seconds at 55ºC, followed by a 1 second extension and data acquisition at 72ºC.
The reasons for primer dimer formation in a NTC are often due to multiple factors. These include: sub-optimal primer annealing temperature (often due to differences in buffering conditions between different qPCR kits), sub-optimal primer synthesis (HPLC purified primers result in less primer dimer formation and are useful for low copy number detection), and poor primer design (using software such as Primer3 is recommended in primer design).
Reducing the total number of cycles in a qPCR reaction is an alternative method if amplification of the primer dimer lies outside of the range of the experimental data. For example, if the sample being interrogated has a Cg of 28 cycles, contains specific product only (no primer dimer), and the NTC amplifies a primer dimer after 37 cycles, the reaction can be run for 35 cycles, rather than 40. This approach can be taken only when the sample being tested gives rise to specific product and not a combination of specific product and primer dimer.
All kit components should be stored at -20ºC. Extra care should be taken when handling the 50X KAPA RT Mix. The RT Mix should always be stored at -20ºC as the enzymes are not thermostable. The KAPA SYBR FAST qPCR Master Mix is sensitive to light and should be protected from direct light particularly during storage. Care should be taken to avoid freeze-thaw cycles. When stored under these conditions and handled correctly, full activity of the kit is retained as indicated on the kit label.