Can I use the Universal Prokaryotic RNA-Seq kit with RNA from any organism?
This system has been designed specifically for prokaryotes. Performance with other organisms may vary.
What methods do you recommend for RNA isolation?
We recommend a column-based method, including:
- Norgen Biotek Total RNA, Soil RNA, or Stool RNA Purification Kits
- Zymo Research QuickRNA™ Kit, ZR Fungal/Bacterial RNA MicroPrep™, or ZR Soil/Fecal RNA MicroPrep™
- 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.
Will the use of RNA purification columns impact my data?
We have observed changes in alignment metrics and expression profiles with the use of purification columns such as the QIAGEN RNeasy column. We recommend consulting the manufacturer to ensure the RNAs of interest are retained after purification.
Do I need to use high-quality total RNA?
The Universal Prokaryotic RNA-Seq kits are 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 yields and may encounter affected sequencing metrics.
Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with the Universal Prokaryotic RNA-Seq?
The system is designed to use total RNA as input. This system utilizes Tecan's customizable AnyDeplete technology to deplete targeted transcripts. rRNA depletion or poly(A) enrichment is not necessary.
How much total RNA do I need for amplification?
The selective priming process is designed to deplete rRNA from 100–500 ng total RNA input.
Can contaminating genomic DNA interfere with the Universal Prokaryotic RNA-Seq kit performance?
Yes, contaminating genomic DNA may be incorporated into libraries. For this reason we recommend DNase treatment during RNA purification. For an explanation of DNase requirements see section III.A. For DNase treatment of RNA samples, refer to Appendix A for guidelines.