> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/rna-library-prep/library-preparation-rna-library-prep-faq-list/000009891.md). # Input recommendations for the Illumina miRNA prep kit **How much RNA input is recommended?** * **Cells and tissues:** Recommended to use 100 ng of total RNA isolated from cell and tissue samples. 1 - 500 ng of total RNA is supported by the kit. * **Serum/Plasma:** Use 5 µl of RNA eluate when 200 µl of serum/plasma has been processed with the miRNeasy Serum/Plasma kit or miRNAeasy Serum/Plasma Advanced kit. * **Exosomes**: Use 5 µl of RNA eluate when 1 ml of serum/plasma has been processed with the exoRNeasy kits for exosome samples. * Note: most column-based RNA isolation kits lose RNA < 200 nt. Labs must use an RNA isolation kit or method that preserves miRNA ( > 15 nt). **Which miRNA isolation kits are recommended?**
KitCatalog #Starting Material
miRNeasy Serum/Plasma kitQiagen 217184Animal and human plasma and serum
miRNeasy Serum/Plasma Advanced kitQiagen 217204Animal and human plasma and serum
exoRNeasy Midi kitQiagen 77144Animal and human plasma and serum
exoRNeasy Maxi kitQiagen 77164Animal and human plasma and serum
* For cells and tissue samples, consider commercially available RNA isolation kits as long as the kit preserves miRNA (> 15 nt). **How is the total RNA input QC'd? Is there a RIN or DV200 threshold?** * Total RNA is recommended to have a RIN > 8, though successful miRNA library prep may still be possible with samples with lower RIN values, as miRNAs are generally protected from degradation due to their small size. Increase the sequencing reads allocated per sample to allow for RNA degradation products. In a total RNA trace, small RNA may appear as a peak of 15 - 150 nt, with miRNA in the 20-30 nt range. * In serum/plasma samples, small RNAs are not visible nor quantifiable. Therefore, quality checks are not recommended. **Can purified miRNA be used as input instead of total RNA? If yes**, **are any protocol modifications needed?** * It is not necessary to enrich for small RNA. If, however, it is desired to work with small RNA samples, small RNA represents approximately 10% of total RNA amounts, so follow the protocol based on the calculated relative total RNA amount. As an example, 100 ng of total RNA translates to 10 ng small RNA; as a result, users would use the adapter and RT primer dilutions recommended for 100 ng total RNA when working with 10 ng small RNA. **Are there known inhibitors to this library prep?** * Samples high in EDTA. EDTA can be removed from input with a bead cleanup or column purification. * Heparin (common in blood samples) inhibits reverse transcription. One option is to treat the total RNA samples with heparinase prior to library construction with the Illumina miRNA prep kit. Note: heparin is retained in a bead cleanup or column purification of the sample, so heparinase must be used. **Is there a recommended positive control sample?** * The XpressRef Universal Total RNA contains both small and large RNA (Qiagen cat. 338112 (human), 338114 (mouse), 338116 (rat)), and can be used as a positive control for this kit. * Spike-in controls can also be added to individual samples, and are often used for biofluids. This allows for monitoring the sample from start to finish and assessing the relative performance of all the samples. Note that samples with spike-in controls will require additional sequencing reads. See manufacturer documentation for details. * Example spike-in controls: * [QIAseq miRNA Library QC Spike-ins](https://www.qiagen.com/us/products/discovery-and-translational-research/next-generation-sequencing/rna-sequencing/mirna-small-rnaseq/qiaseq-mirna-library-qc-pcr-assays-arrays) * [TamiRNA miND Spike-In](https://www.tamirna.com/mind-spike-in-controls-for-small-rna-sequencing-tamirna/) \ \ \
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