Input recommendations for the Illumina miRNA prep kit
How much RNA input is recommended?
Cells and tissues: Recommend 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. Customers must use an RNA isolation kit or method that preserves miRNA ( > 15 nt).
Which miRNA isolation kits are recommended?
Kit
Catalog #
Starting Material
miRNeasy Serum/Plasma kit
Qiagen 217184
Animal and human plasma and serum
miRNeasy Serum/Plasma Advanced kit
Qiagen 217204
Animal and human plasma and serum
exoRNeasy Midi kit
Qiagen 77144
Animal and human plasma and serum
exoRNeasy Maxi kit
Qiagen 77164
Animal and human plasma and serum
For cells and tissue samples, customers should be able to use 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 cutoff or DV200?
Total RNA is recommended to have a RIN > 8, though successful miRNA library prep is still possible with samples whose RIN values are lower, as miRNAs are generally protected from degradation due to their small size. In a total RNA trace, small RNA may appear as a peak of 15 - 150 bases, with miRNA in the 20-30 bases 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, what protocol deviations to make?
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. Therefore, 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 that customers can use?
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 being able to assess the relative performance of all the samples. Note that samples with spike-in controls will require additional sequencing reads. Please see manufacturer for details.
Example spike-in controls:
For any feedback or questions regarding this article (Illumina Knowledge Article #9891), contact Illumina Technical Support [email protected].
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