What methods do you recommend for RNA isolation?
We recommend a column-based method, including:
- Norgen Biotek Total RNA Purification Kit
- Zymo Research Quick-RNA™ Kits
- Arcturus PicoPure® RNA Isolation Kit
- Ambion PureLink® RNA Mini Kit
- Qiagen RNeasy Kits
Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
Can I use TRIzol® or other phenol-chloroform based extractions for RNA isolation?
We do not recommend the use of TRIzol or similar methods as any carryover of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.
Can I use carrier RNA during RNA isolation?
We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Tecan NGS Technical Support for more information.
Do I need to use high quality total RNA?
Yes. Use of lower quality RNA may result in poor performance. One approach to determining RNA quality is the Agilent Bioanalyzer’s RNA Integrity Number (RIN). Clean RNA with a RIN score greater than 7 should amplify well.
How much total RNA input do I need for amplification?
We recommend staying within the specified range of 5 to 100 ng of total RNA starting material. We often suggest 20 ng input as an appropriate starting point. Input greater than 100 ng may adversely affect amplification.
Can I use a total RNA input of less than 5 ng?
The Ovation RNA Amplification System V2 has been validated for total RNA input amounts of 5 to 100 ng. Using input quantities outside the recommended range will affect cDNA yields.
Can I omit quantification of input RNA?
It is important that RNA input amounts be kept within the 5 to 100 ng range recommended; therefore, we do not recommend omitting quantification of input RNA.
Can I use mRNA instead of total RNA as starting material?
Purified poly(A) RNA has been successfully used as input to the Ovation RNA Amplification System V2. It may be necessary to reduce the input of mRNA to a level comparable to the mRNA present in 5 ng to 100 ng of total RNA.
Can the Ovation RNA Amplification System V2 kits be used for amplification of DNA?
The Ovation RNA Amplification System V2 is designed to amplify mRNA, not DNA. Please contact Tecan NGS Technical Support for more information on our SPIA-based DNA amplification product offerings.
Can I use the Ovation RNA Amplification System V2 on prokaryotic RNA samples?
The Ovation RNA Amplification System V2 relies on the presence of a poly(A) tail for priming. Therefore, it will not amplify most prokaryotic RNA.
Are there any tissues that will not work with the Ovation RNA Amplification System V2?
We have not encountered any good-quality, clean RNA samples containing poly(A) RNA that will not work with the Ovation RNA Amplification System V2.