Q3. 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.
Q4. 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 solvent 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.
Q5. Can I use carrier RNA during RNA isolation?
No. Residual carrier RNA can result in poor data quality.
Q6. How much total RNA do I need for library generation?
Revelo mRNA-Seq for MagicPrep NGS can be used with 10 ng to 1 μg purified, high-quality (RIN ≥7.0) total RNA. Input amounts outside this range may affect reaction stoichiometry, resulting in sub-optimal libraries that produce variable and unsatisfactory results. Lower input amounts will potentially result in insufficient yields depending on the requirements of the analytical platform.
Q7. Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with Revelo mRNA-Seq for MagicPrep NGS?
No rRNA depletion or poly(A) enrichment step is needed. Use total RNA inputs with Revelo mRNA-Seq for MagicPrep NGS.
Q8. Do you recommend DNase treatment of purified total RNA samples?
Yes. When using purified total RNA samples, large amounts of contaminating genomic DNA may amplify during the process. For this reason DNase treatment is recommended prior to running Revelo mRNA-Seq for MagicPrep NGS.
Q9. Can I use Revelo mRNA-Seq for MagicPrep NGS with RNA from any organism?
Revelo mRNA-Seq for MagicPrep NGS has been designed and tested only with total RNA inputs isolated from human samples. However, any input RNA sample containing some fraction of 3ˇ-poly(A) sequence should yield a sequenceable library. For more information, contact Tecan NGS Technical Support at firstname.lastname@example.org.