Ambion - Designing a Successful qRT-PCR Experiment

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mRNA Detection Sensitivity by Real-time RT-PCR Comparing Ambion's MessageSensor™ RT/SuperTaq™ Real-time with Invitrogen's SuperScript™ One-Step RT-PCR with Platinum™ Taq

Target	Input Total RNA	C_t MessageSensor RT/SuperTaq Real-time	C_t SuperScript II/Platinum Taq	delta C_t Improvement with MessageSensor	RT Reaction Temperature
GAPDH	1 pg	32.57	34.48	1.91	44
	100 fg	36.57*	40		44
ß-actin	100 pg	29.96	29.78	-0.17	42
	10 pg	33.69	40	6.31	42
TERT	1 ng	31.94	34.18	2.25	42
	100 pg	36.98	40	3.02	42
cdc-2	100 pg	30.06	34.33	4.27	42
	10 pg	33.63	40	6.37	42
C-JUN	10 ng	27.74	40	12.27	42
	1 ng	31.46	40	8.55	42

C-JUN	10 ng	28.9	40	11.1	50
	1 ng	32.88	40	7.13	50
MYB	100 ng	25.99	32.22	6.23	50
	10 ng	30.07	40	9.94	50
GADPH	10 pg	29.9	31.9	2	50
	1 pg	33.31	35.8	2.49	50
cdc-2	100 pg	30.21	33.34	3.13	50
	10 pg	33.71	40	6.3	50
ß-actin	100 pg	32.02	33.17	1.16	50
	10 pg	34.79	36.86	2.07	50

Table values are the average of duplicate data points at amounts of input total RNA that are known to be at or near the detection limit. An undetectable signal is recorded as a cycle threshold (C_t) of "40.00". In cases where one of the two replicates was undetectable (C_t>40), the remaining detectable signal (and corresponding C_t) is marked with an asterisk (*). The column "delta C_t, Improvement with MessageSensor" represents the decrease in cycle threshold (that is, superior detection sensitivity) using the MessageSensor RT/SuperTaqþ compared to the SuperScript II/Platinum Taq Kit. In cases where the SuperScript II/Platinum Taq failed to give a signal, the C_t shift is greater than or equal to the value given. Note that delta C_t = 3.3 is equivalent to a 10-fold difference in detection sensitivity.

qRT-PCR reactions were performed according to the manufacturer's instructions. All mRNA targets were human sequences, and the source of RNA was Ambion's HeLa S3 Total RNA. Briefly, a 25 µl one-step real-time RT-PCR reaction was assembled using TaqManþ probes, and the data analyzed using an ABI 7900 instrument. For all targets, the determination of cycle threshold was made at same fluorescent signal intensity (delta Rn).