TruSeq Small RNA input best practices

Best Practices

• This kit is designed to start with total RNA (although pre-purified miRNA can be used). It is critical that this total RNA be high-quality, with a RIN of at least 8. Any standard RNA isolation technique can be used, as long as it retains miRNA (for example, some spin column RNA purifications will not capture the miRNA fraction). Use of mildly degraded RNA will result in low yields and loss of the low end of the dynamic range of the assay. More severely degraded total RNA will most likely produce low or no yield.

• The total RNA is suspended in nuclease-free water or 10 mM Tris. DEPC will not interfere with the enzymes in this kit.

• This kit is designed for use with 1 µg of total RNA. Illumina does not support starting material amounts lower than 1 µg.

What is the minimum input material? The protocol is optimized using 1.0 µg of total RNA.

Can this protocol use purified small RNA as starting material? Yes. Note that there can be high sample variability using some commercially available small RNA spin column purification methods, and validation of any purification method may be necessary. If purified small RNA is used as starting material, 10-50 ng should be used.

Does the TruSeq small RNA protocol work with plant small RNA? Some plant small RNA molecules are methylated at their 3' end. The RNA ligase used is somewhat able to ligate to several common methyl modifications with sufficient efficiency to support library construction, though efficiency may be lower.

For any feedback or questions regarding this article (Illumina Knowledge Article #1397), contact Illumina Technical Support [e-mail].