Can I do a One-Step Real-Time (RT-PCR) reaction with TaqMan® Gene Expression Assays?
While a one-step RT-PCR reaction is compatible with TaqMan® Gene Expression Assays, they have not been optimized for use in a one-step reaction. TaqMan® Gene Expression Assays have been designed, tested, and optimized using Applied Biosystems® Universal Conditions for a two-step RT-PCR reaction. It is recommended to use the SuperScript® VILO™cDNA Synthesis Kit (P/N 4453650, 4453651) for converting your RNA to cDNA. Other RT kits that can be used are the High Capacity RNA-to-cDNA kit and High Capacity RNA-to-cDNA Master Mix. If your starting material is cell culture, you can use Cells-to-cDNA™ II. After the reverse transcription, you can use TaqMan® Gene Expression Master Mix, TaqMan® Universal PCR Master Mix, with or without Amperase UNG to run the PCR.
If you need to do One-Step RT-PCR, it is recommended to use TaqMan® RNA-to CT™ 1-Step kit (PN 4392653). If the starting material is cell culture, TaqMan® Gene Expression Cells-to-CT™ is recommended.
Answer Id: 1835
Can cDNA be generated using the High Capacity cDNA Reverse Transcription kit (SKU 4322171) for quantitative qPCR applications?
Yes, the single-stranded cDNA generated via the High-Capacity cDNA Reverse Transcription can be used in real-time PCR applications. In addition, we also recommend Superscript® VILO™ cDNA Synthesis Kit, High Capacity RNA to cDNA kit, and/or High Capacity RNA to cDNA Master Mix for total RNA.
Answer Id: 2149