Reality Reverse Transcriptase is a modified version of M-MLV reverse transcriptase with increased thermal stability and deactivated RNase H activity. In combination with optimized buffers and reagents, Reality Reverse Transcriptase synthesizes complementary DNA from single-stranded RNA or DNA templates. The enzyme is sensitive, specific and capable of synthesizing highly-structured and long cDNA fragments. The cDNA produced is appropriate for both PCR and qPCR analysis. Reality offers higher cDNA yields than some leading competitors with highly sensitive efficient synthesis from low-abundance RNA transcripts. Suitable for cDNA Synthesis of products from 100bp up to 10kb. The kit contains Reality Reverse Transcriptase (200U/µl), 5X buffer, dNTP mix (10mM), DTT solution (100mM), RNase inhibitor (40U/µl), Oligo dT 20mer primer (100µM), random hexamers (100µM), positive control RNA (10ng/µl), and RNase-free water.

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

Step 1A: cDNA synthesis without denaturation

Component	Stock Concentration	Final Concentration	Volume
RNA Template	-	Total RNA: 10pg-5µg or mRNA: 10pg-500ng	X µl
Primer	100µM	Gene Specific Primer: 10-20pmol (50-100ng) Oligo dT or random hexamer: 50pmol	0.1-0.2µl 0.5µl
dNTP Mix	10mM	500µM	1µl
DTT Solution	100mM	5mM	1µl
RNase Inhibitor	40U/µl	40U	1µl
Reality Reverse Transcriptase	200U/µl	100U	0.5µl
5X Buffer	5X	1X	4µl
RNase Free Water	-	-	QS to 20µl

Step 1B (Optional): cDNA synthesis with denaturation
First, prepare the Template/Primer mix using the table below and incubate 5min at 65-70°C.

Component	Stock Concentration	Final Concentration	Volume
RNA Template	-	Total RNA: 10pg-5µg or mRNA: 10pg-500ng	X µl
Primer	100µM	Gene Specific Primer: 10-20pmol (50-100ng) Oligo dT or random hexamer: 50pmol	0.1-0.2µl 0.5µl
RNase Free Water	-	-	QS to 10µl

Second, prepare the Reaction mix using the table below.

Component	Stock Concentration	Final Concentration	Volume
dNTP Mix	10mM	500µM	1µl
DTT Solution*	100mM	5mM	1µl
RNase Inhibitor**	40U/µl	40U	1µl
Reality Reverse Transcriptase***	200U/µl	100U	0.5µl
5X Buffer	5X	1X	4µl
RNase Free Water	-	-	QS to 10µl

*Adding up to 5mM DTT may increase the yield and is recommended for individual optimization.
**An additional 20-40 Units of RNase inhibitor per reaction is recommended when using low amounts of starting RNA.
***Increased transcription levels may be achieved by increasing the amount of enzyme to 200 Units.

Third, add 10µl of Reaction mix to 10µl of Template/Primer mix and pipette gently up and down on ice.

Step 2: Incubation
Gene Specific Primers: 30-60min at 50°C.
Oligo dT or random hexamer: 10min at 42°C followed by 30-60min at 50°C.

Step 3 (Optional): Heat inactivation
Heat the mixture for 10min at 70°C to inactivate Reverse Transcriptase.

Step 4 (Optional): RNA removal
Add 2 Units of DNase-free RNase and incubate for 20min at 37°C. The cDNA can now be used as a template in PCR and should be stored at -20°C.