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.
Can I use Universal RNA-Seq with NuQuant with RNA from any organism?
This system has been designed for use with a broad range of different organisms using organism-specific probes to target specific transcripts for depletion. AnyDeplete probe mixes for human (rRNA), mouse (rRNA plus globin) and Drosophila (rRNA) are available. Please contact Tecan NGS Technical Support for a list of available custom AnyDeplete designs or to design a new custom AnyDeplete probe mix.
Do I need to use high-quality total RNA?
The Universal RNA-Seq with NuQuant kit 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 increasing the amount of total RNA input to 100-250 ng. With highly degraded samples, users may experience lower yields and may encounter affected sequencing metrics.
Do I need to perform an rRNA depletion, poly(A) enrichment step, or globin depletion before processing total RNA samples with the Universal RNA-Seq with NuQuant kit?
No. The system is designed to use total RNA as input. rRNA depletion or poly(A) enrichment are not necessary. rRNA, as well as other transcript types, can be targeted for depletion using the AnyDeplete technology integrated in the workflow. Custom globin depletion is available for several mammalian species. Contact Tecan NGS Technical Support for more information.
How much total RNA do I need for library generation?
We recommend 10-100 ng high quality RNA, 100-250 ng degraded of FFPE RNA, or 100-500 ng whole blood RNA as input into the kit. Please refer to section III.A of the User Guide for a discussion of RNA input requirements.
Can contaminating genomic DNA interfere with the Universal RNA-Seq with NuQuant performance?
Yes, contaminating genomic DNA can interfere with accurate RNA quantification and may be incorporated into libraries. For this reason we recommend DNase treatment during RNA purification.
For samples that have not undergone a thorough DNase treatment during RNA purification or is suspected of containing contaminating genomic DNA, we recommend starting the protocol at section V.A. First Strand cDNA Synthesis with Integrated DNase Treatment.
For an explanation of DNase requirements see section III.A of the User Guide.