Can I do a One-Step Real-Time (RT-PCR) reaction with TaqMan® Gene Expression Assays?

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Answer

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: E1835

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da73b048befec3f0ef20c8252ff3803a_FAQ

Can cDNA be generated using the High Capacity cDNA Reverse Transcription kit (SKU 4322171) for quantitative qPCR applications?

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Answer

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: E2149

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e07e9550b811db83feaf7e970b1d5a92_FAQ

What type of primers are in the High-Capacity RNA-to-cDNA™ Kit?

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Answer

The High Capacity RNA-to-cDNA™ Kit includes a 2X RT buffer mix, which is composed of dNTPs, random octamers, and oligo(dT)16.

Answer Id: E7473

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65f09faf6cfcb6b6bdf1f16e1d6ced6d_FAQ

What is the difference between the High-Capacity RNA-to-cDNA™ Kit, the High-Capacity cDNA Reverse Transcription Kit, and the SuperScript® VILO™ Master Mix?

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Answer

The main differences are in the enzyme and type of reverse transcription primer used. The High-Capacity kits use MultiScribe™ Reverse Transcriptase, while the SuperScript® VILO™ Master Mix uses SuperScript® III Reverse Transcriptase. The High-Capacity RNA-to-cDNA™ Kit uses a blend of random primers and oligo(dT), while the High-Capacity cDNA Reverse Transcription Kit and the SuperScript® VILO™ Master Mix use only random primers.

Answer Id: E7474

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a8cc54f23e71d0658169ef41b0a4e954_FAQ

What reverse transcription kit should I use for my gene expression experiment?

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Answer

Please review this selection table (https://www.lifetechnologies.com/us/en/home/life-science/pcr/reverse-transcription/cdna-synthesis-kits/rt-real-time-pcr.html) to help choose the best reverse transcription kit for your needs.

Answer Id: E7475

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f6ef1d7df8e906925a3c0f2200240b22_FAQ

How much RNA do I need for the High-Capacity cDNA Reverse Transcription Kit?

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Answer

With the High Capacity Reverse Transcription Kit, you can use from 20 ng up to 2 μg of total RNA in a 20 μL reaction.

Answer Id: E7476

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45ad3c08c0bd54c8bb973d306f393449_FAQ

Can I order the RNase Inhibitor from the High-Capacity cDNA Reverse Transcription Kit separately?

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Answer

Yes. You can order the RNase Inhibitor separately as Cat. No. N8080119.

Answer Id: E7477

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0c7acac063de2986d7ff88662e3489c2_FAQ

Can I order the random primers from the High-Capacity cDNA Reverse Transcription Kit separately?

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Answer

No. The 10X random primers are only available as part of the High-Capacity Reverse Transcription Kit.

Answer Id: E7478

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6fbee7f9ce9f3905661368be3e2e7109_FAQ

How long can I store the cDNA from my reverse transcription step?

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Answer

You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.

Answer Id: E7479

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959307e8fc5aff3b59bbd2013cd9e56e_FAQ