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Description:
The RT-PCR Primer and Control Set is provided for use as a positive control in RT-PCR applications. It contains human β-actin sense and anti-sense primers (control primers) and total HeLa RNA sufficient for 20 amplification
reactions of 50 μl each. The control primers are designed from the human β-actin mRNA gene and produce a 353-bp RT-PCR product (1).

Quality Control:
The product is tested functionally using 10 pg of total HeLa RNA as the template for amplification of a 353-bp segment of β-actin mRNA (40 cycles).A minimum of 25 ng of the RT-PCR product was obtained under these
conditions.
Limited Label License No. 4:
This product is optimized for use in the Polymerase Chain Reaction (PCR) covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche, Ltd. (“Roche”). No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed
suppliers such as Invitrogen, when used in conjunction with an Authorized Thermal Cycler, or is available from Applied Biosystems. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc.,1145 Atlantic Avenue, Alameda, California 94501.

Procedure:
This protocol uses components from the SuperScript One-Step RT-PCR System with Platinum® Taq (Cat. No. 10928-034 or 10928-046).
1. Serially dilute the Total HeLa RNA in distilled water to 1 pg-10 ng per 5 μl.
2. Add the following components to a sterile 0.5-ml microcentrifuge tube sitting on ice:
Note: The absence of genomic DNA in RNA preparation can be verified by omitting the RT/Platinum® Taq Mix and substituting 2 units of Platinum® Taq DNA polymerase in the reaction.

3. Gently mix and centrifuge briefly to collect the contents to the bottom.
4. Depending on the thermal cycler used, overlay with silicone oil, if necessary.
Reference:
1. Lee, E.H., Sitaraman, K., Schuster, D., Rashichian, A., (1997) Focus 19, 2.
5. Thermal cycler program:
Note: The following cycling conditions were established using a DNA Thermal Cycler 9600 or 2400 (Perkin-Elmer) and may have to be altered for other thermal cyclers. Efficient cDNA synthesis can be achieved in a 30-min incubation at 50°C.

∗ For use in Perkin-Elmer Model 480 cycler, use 30 s denaturation instead of 15 s.
6. Analyze 10 μl of the RT-PCR products on a 1.5% agarose gel.
7. A 353-bp RT-PCR product is clearly visible by UV transillumination of an ethidium bromide stain gel starting with 10 pg of total HeLa RNA.