Input Requirements

Input Requirements

The TruSeq Small RNA Library Prep protocol is optimized for 1 µg of high-quality human or mouse brain total RNA as input.

Total RNA Input

Small RNA populations can vary significantly between different tissue types and species. Use of RNA from other species, tissues, or qualities may require further optimization with regard to the initial input amount and selection of the desired bands during the final gel excision. The types and coverage of small RNAs sequenced will also vary depending on which bands are selected during gel excision.

It is very important to use high-quality RNA as the starting material. Use of degraded RNA can result in low yield or failure of the protocol. Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than 8.

Alternatively, you can run a formaldehyde 1% agarose gel and judge the integrity of RNA upon staining with ethidium bromide. High quality RNA shows a 28S rRNA band at 4.5 kb that should be twice the intensity of the 18S rRNA band at 1.9 kb. Both kb determinations are relative to a RNA 6000 ladder. The mRNA will appear as a smear from 0.5–12 kb.

Purified mRNA Input

You can also use previously isolated micro RNA as starting material. Use the entire fraction of small RNA purified from 1–10 µg of total RNA. Fewer undesired bands will be seen during the subsequent gel extraction using this method. Purified small RNAs must be in molecular grade water or 10 mM Tris-HCI, pH 8.5.

Positive Control

Illumina recommends using Ambion FirstChoice human brain total RNA (catalog # AM7962) as a positive control sample for this protocol. This preparation is certified to contain the small RNA fraction.