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
For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:
- Norgen Biotek FFPE RNA Purification Kit
- Zymo Research Quick-RNA™ FFPE Kit
- Arcturus® Paradise® PLUS FFPE RNA Isolation Kit
- PureLink™ FFPE RNA Isolation Kit
- Qiagen RNeasy FFPE Kit
Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method. Isolation methods that capture RNAs shorter than 200 nt may result in increased 5S and 5.8S rRNA. Do not use carrier RNA during isolation.
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 library generation?
Trio RNA-Seq can be used with purified total RNA in the range of 500 pg to 50 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 Trio RNA-Seq?
rRNA depletion or Poly(A) enrichment is not required. The input range of 500 pg to 50 ng refers to total RNA.
Can I use poly(A) RNA as an alternative to total RNA?
Trio RNA-Seq has not been tested for use with poly(A) RNA.
Do I need to use high-quality total RNA?
Trio RNA-Seq 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 a wide range of samples. For FFPE or degraded samples, we recommend using total RNA inputs of 10–50 ng. With highly degraded samples, users may experience reduced data quality.
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 I skip DNase treatment if I already incorporated DNase treatment during RNA isolation?
We recommend performing DNase treatment as a part of the Trio RNA-Seq workflow, because residual amounts of genomic DNA can be amplified and impact sequencing results.
Can I use Trio RNA-Seq with RNA from any organism?
Trio RNA-Seq System has been designed for use with total RNA isolated from a broad range of different organisms. The system requires obtaining organism-specific probes to target specific transcripts for depletion. See Table 2 in the User Guide for a list of available probe sets, or contact Tecan NGS Technical Support at firstname.lastname@example.org for more information on designing custom probes.