Superior stability after reaction setup at room temperature
Amplification of GC-rich amplicons
Amplification of long amplicons
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
Optional pipetting control
Superior sensitivity
Superior specificity
OneStep Ahead RT-PCR procedure
HeLa total RNA (10 and 1 pg) was used as a template for amplification of ACTB in triplicates, according to suppliers’ instructions. Reactions were either set up on ice or left at room temperature for the times indicated before analysis on a 2% agarose gel. Distinct, gene-specific bands are observed with the QIAGEN OneStep
Ahead
RT-PCR Kit even after a 2 h incubation at room temperature before cycling (blue arrow), whereas reactions performed with kits from competitors deteriorate as time progresses. Gene-specific bands appear significantly weaker, if present at all, while primer-dimers (red arrows) become more prominent.
HeLa total RNA (100 ng) was used as template for amplification of TNFRI (581 bp) with a GC ratio of 67.1%. Reactions were performed in triplicate, according to the suppliers’ instructions. Green arrows indicate specific product. Q-Solution was used for the QIAGEN reactions.
HeLa total RNA (100 and 10 ng) was used as a template for amplification of RANBP2 (3 kbp).
Potassium ions (K
+
) bind to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH
4
+
, which exists both as ammonium ion and as ammonia under thermal‐cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer–template binding over a wide temperature range.
The kit comes with optional pipetting controls. The Master Mix tracer is an inert orange dye that can be added to the Master Mix. The Template Tracer is an inert blue dye that can be added to the template. When adding the template to the Master Mix, the color turns green, providing a visual indication of correct pipetting. In addition, both dyes allow tracking during gel electrophoresis. The dyes run at about 50 bp (orange) and 4000 bp (blue) on a 1% agarose gel.
Indicated amounts of HeLa total RNA (in pg) were used as template for amplification of GAPDH (831 bp) and ACTB (295 bp) in duplicate, according to the suppliers’ instructions. Green arrows indicate specific product, red arrows indicate primer‐dimers. Analysis was performed using the QIAxcel.
Indicated amounts of HeLa total RNA (in ng) were used as template for amplification of EIF2B4 (107 bp) in duplicate, according to the suppliers’ instructions. Green arrows indicate specific product, red arrows indicate primer‐dimers, purple arrows indicate nonspecific amplification products. Analysis was performed using the QIAxcel.
The QIAGEN OneStep
Ahead
RT-PCR Kit allows fast and easy RT-PCR setup whatever your application - virus detection, molecular diagnostics research or gene expression - just mix all components together in one tube and start your thermal-cycler program. The reaction mixture contains all of the reagents required for both reverse transcription and PCR; nothing needs to be added once the reaction has been started. As a visual pipetting control, you can add the optional orange Master Mix Tracer and blue Template Tracer.