Significantly reduced PCR times.
Wide dynamic range and high sensitivity.
Faster results without compromising sensitivity.
Sensitive two-step RT-PCR.
Detection over 7 Logs of template.
Specific primer annealing.
Fast primer annealing.
QuantiFast Probe Kits reduce total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit).
L
: LightCycler 2.0;
A1
: ABI PRISM 7900;
A2
: Applied Biosystems 7500;
A3
: ABI PRISM 7000.
The QuantiFast Probe PCR +ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93% in two-step PCR on the Mastercycler ep
realplex
. Duplicate reactions (25 μl volume) were performed using 10-fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin (a regulatory protein).
NTC
: no template control.
Reactions were run in duplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan gene expression assay for IL1RN (a cytokine) on the ABI PRISM 7900. The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier A
II
(which was used according to the standard cycling protocol), providing much lower C
T
values and transcript quantification from down to 10 pg cDNA.
Reactions were run in triplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a Primer Express designed TaqMan assay for IL8 (a chemokine) on the ABI PRISM 7900.
[A]
The QuantiFast Kit showed greater sensitivity than
[B]
the kit from Supplier I (which was used according to the standard cycling protocol), providing lower C
T
values and transcript quantification from down to 0.01 ng cDNA.
Duplicate two-step RT-PCR reactions were run using a set of primers and FRET probes for
b
-2 microglobulin from Supplier R on the LightCycler 2.0. The amplification plots for the QuantiFast Kit show the kit's wide dynamic range.
A balanced combination of KCl and (NH
4
)
2
SO
4
promotes specific annealing of primers and probes to the PCR template. K
+
binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH
4
+
destabilizes weak hydrogen bonds between mismatched bases.
[A]
Q-Bond in QuantiFast Buffer increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times.
[B]
Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.