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.
How much total RNA do I need for amplification?
The Ovation RNA‑Seq System V2 can be used with purified total RNA in the range of 500 pg to 100 ng. Input amounts outside this range may produce unsatisfactory and variable results.
Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with the Ovation RNA‑Seq System V2?
rRNA depletion or Poly(A) enrichment is not required. The input range of 500 pg to 100 ng refers to total RNA.
Can I use poly(A) RNA as an alternative to total RNA?
Yes, if you are primarily interested in sequencing reads from mature coding transcripts then poly(A) RNA may be substituted for total RNA. Tecan has demonstrated that poly(A) RNA can be isolated from 5 to 500 ng of total RNA and used as input into the Ovation RNA-Seq System V2 protocol with good results. We recommend using at least 500 pg of isolated poly(A) RNA.
Can DNA be used as input for the Ovation RNA-Seq System V2?
No. The Ovation RNA-Seq System V2 is designed to amplify RNA, not DNA.
Do I need to use high-quality total RNA?
The Ovation RNA-Seq System V2 is designed to work with purified total RNA. When using purified total RNA, samples should be of high molecular weight with little or no evidence of degradation. While it is impossible to guarantee the highest levels of performance when using RNA of lower quality, this system should allow the successful analysis of somewhat degraded samples. With such samples, users may experience lower amplification yields and should plan to use relatively higher RNA inputs.
Do you recommend DNase treatment of purified total RNA samples?
Yes. When using purified total RNA samples, contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.
Can RNA from any organism be used in the Ovation RNA-Seq System V2?
The Ovation RNA-Seq System V2 has been optimized for use with mammalian samples. The system should work with most non-mammalian samples, however, you may observe a higher percentage of rRNA than is seen with mammalian samples.