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. NH4+, 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
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
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.