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 carry over 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 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 RNA input of 5–100ng RNA from whole blood or total cellular RNA; however, due to the high levels of globin present in whole blood RNA samples, we advise using inputs of 20 ng or higher when feasible.
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 DNA be used as input for the Ovation RNA Amplification System V2?
No. The Ovation RNA Amplification System V2 is designed to use total RNA as input.
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.
Do you recommend any specific whole blood RNA isolation methods?
We do not specifically require one method of RNA preparation, however, for whole blood samples the QIAGEN PAXgene products for stabilization and RNA isolation have been validated and are compatible with the Ovation Whole Blood Solution. In general, any method that yields high quality, non-degraded RNA that is free of organic solvents and contaminants should work well.
Can the Encore Biotin Module be used for fragmentation and labeling of RNA?