The RealCount One Step RT-qPCR Kit with UNG is ideal for accurate quantification of RNA or DNA in a variety of samples such as whole blood, serum, plasma, urine, saliva, cerebral spinal fluid, feces, and plant. The kit can be used for intercalating dye and probe-based chemistries on a variety of qPCR instruments. Aptamer modified warm start Reality Reverse Transcriptase synthesizes complementary DNA (cDNA) from single-stranded RNA or DNA templates. PCR amplification of the cDNA by TaqTivate Hot Start Taq is monitored in real time. The 4X master mix formulation contains inhibitor reducing buffer components optimized for mammalian tissue samples. Uracil-DNA Glycosylase (UNG) and dUTP are included in the master mix to reduce potential false positive results from carry over contamination. The kit contains Reality Reverse Transcriptase (200U/µl), 4X TaqTivate RT-qPCR Master Mix, DTT solution (100mM), 20X TealReveal Dye, and ROX passive fluorescent reference dye (25µM).

Protocol: The following setup is recommended for a 20µl reaction.

Table 1: Single Step Reaction Setup

Component	Volume	Final Concentration
TaqTivate Hot Start Master Mix, 4X	5µl	1X
Reality Reverse Transcriptase, 200U/µl*	0.5µl	5U/µl
DTT, 100mM*	1µl	5mM
Target Specific Primers and Probe	1µl	1X
Sample	Xµl	1pg-100ng
TealReveal Dye, 20X (if required)	1µl	1X
ROX Reference Dye, 25µM (if required)	Xµl	See Table 2
Nuclease Free Water	QS to 20µl	-

*Reactions must be placed in cycler immediately following completion to maintain the integrity of the Reverse Transcriptase and DTT.

Table 2: Recommended ROX Concentrations

Type	Instrument		ROX Final Concentration
	Manufacturer	Instrument Name
No ROX	Roche	LightCycler 480, LightCycler 2.0	None
	BioRad	iCycler, MyiQ, MiQ 2, iQ 5, CFX-96, CFX-384, Chromo4, MJ Opticon, Option2, MiniOpticon
	Qiagen	Roto-Gene Q, Roto-Gene 3000, Roto-Gene 6000
	Illumina	Eco RealTime PCR System
	Eppendorf	Mastercycler realplex
	Cepheid	Smart Cycler
Low ROX	ABI	7500, 7500 Fast	30nM
	Stratagene	MX4000P, MX3000P, MX3005P
High ROX	ABI	5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne plus, Viia7	300nM

Table 3: Recommended Cycling Conditions

Stage	Cycling Step	Cycles	Temperature	Holding Time
1	UNG Incubation	1	25°C	2min
2	RT Incubation	1	55°C	15min
3	Enzyme Activation	1	95°C	2min
4	Amplification**	40	95°C	10sec
			60°C	60sec

**Temperature and holding times will depend on primer set used.