Description:
RT-PCR is widely used for measuring gene expression in tissue samples or cell culture systems. Traditionally, it is performed in two separate reaction steps. First-strand cDNA is reverse-transcribed from total RNA or poly (A)+ RNA using a reverse transcriptase. Next, the cDNA is amplified by PCR using a DNA polymerase in another reaction.

The MCLAB's HoTaq Taqman probe One-step Real-time RT-PCR Kit offers a unique system for performing probe based realtime RT-PCR in a single step within a single tube with an optimized reaction condition, which utilizes our own proprietary engineered QuantumScript HD reverse transcriptase and hot-start Taq DNA Polymerase with high purity . No additional reagents or steps are required once the reaction is initialized. This novel kit enables to quantitatively detect specific RNA targets with high sensitivity, unparalleled convenience, and a wide dynamic range.

The technique reduces the risk of cross-contamination and minimizes the use of reagents. This method is particularly useful for applications in which the expression of a small number of genes that must be analyzed in many different total RNA samples, and robustly amplifing high-abundance transcripts from crude total RNA preparations.

Applications:

• Gene Expression Analysis
• Genotyping
• Real-Time PCR

Primer and probe design:
To achieve the best performance, appropriate software, such as ABI Primer Express^TM, should be used.

Recommended Reaction Conditions:
One cycle at 50°C for 10 to 30 minutes ^*;
One cycle at 95°C for 10 minutes;
Followed by 40 cycles of: 95°C for 15 seconds, 60°C for 1 minute ^#;
4°C hold (optional)
^* Reaction time could be adjusted according to RNA input.
^# Cycle number and annealing temperature are experiment dependent.

Recommended Storage Condition: -20°C

Notes:
To achieve accurate quantification, it is highly recommended to (1) avoid any RNase contamination; (2) design probe on sense strand; (3) use primer concentration from 100nM to 300nM; (4) shorten time between setting up reaction and loading plate onto PCR machine.

Advantages:
One-step RT-PCR products are faster.

List of Components:

Sufficient reagents are supplied in the MCLAB's HoTaq One-step Real-time RT-PCR Kit for 40 or 200 rxns. Upon receipt of the kit, immediately store the components at –20 °C in a freezer without a defrost cycle. It is recommended to prevent light exposure as little as possible.

40 rxns:

2x RT-PCR Buffer*: 1.5ml x 1

25x RT-PCR Enzyme Mix: 40 ul x1

200 rxns:

2x RT-PCR Buffer*: 7.5ml x 1

25x RT-PCR Enzyme Mix: 200 ul x1

* Contains regular level of ROX, dNTPs and optimized buffer components for Real-time PCR machines ABI 7000, 7300, 7700, 7900 and Stratagene Mx 3000P, Mx 3005P.

Additional  Materials Required:

The following reagents, instruments and consumables are supplied by the user:

• Template RNA
• Gene-specific primers and probe
• DEPC-treated water
• Microcentrifuge
• Real-time thermal cycler
• PCR tubes/plates

Reference:
1. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. 1991. Proceedings of the National Academy of Sciences USA 88:7276-7280.
2. Livak, K. J., Flood, S. J. A., Marmaro, J., Giusti, W., and Deetz, K. 1995. PCR Methods and Applications 4:357-362.
3. Lee, L. G., Connell, C. R., and Bloch, W. 1993 Nucleic AcidsResearch 21:3761- 3766.