Input Recommendations

The Illumina miRNA Prep kit is optimized to prepare miRNA and other similarly sized RNAs with a 3' hydroxyl group and a 5' phosphate group (such as piRNA) sequencing libraries for use with Illumina sequencing systems. RNA molecules that are 50 bp or smaller and have a 3' hydroxyl and 5' phosphate are included in the library.

Total RNA containing miRNA is the required starting material for the Illumina miRNA Prep kit. It is not necessary to enrich for small RNA.

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Quantify input RNA per the following recommendations:

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Successful library preparation depends on accurate quantification of input RNA. Use multiple methods to verify results.

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Use fluorometric-based methods, such as Qubit, for accurate quantification of input RNA. UV spectrophotometric-based methods measure any nucleotides present in the sample, including gDNA, dsDNA, ssDNA, and free nucleotides, which can give an inaccurate measurement of RNA.

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Quantification methods depend on accurate pipetting methods. Make sure that the pipettes are calibrated. Avoid using pipettes at the extremes of volume specifications.

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Assess RNA quality per the following recommendations:

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The 260/280 nm absorbance ratio is used as an indication of sample purity. Values from 1.9 through 2.1 indicate relatively pure RNA. The presence of DNA or small nucleic acid fragments, such as nucleotides, can compromise both absorbance measurements.

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The spectral properties of nucleic acids depend on pH values. Illumina recommends preparing dilutions and measuring absorbance in 10 mM Tris-HCl, pH 7.5.

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It is not useful to assess the concentration and purity of total RNA derived from fluids and/or exosomes.

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Make sure that samples are free of contaminants.

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Assess RNA integrity per the following recommendations:

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Confirm the integrity and size distribution of total RNA from cells and fresh or frozen tissue by using an automated analysis system. Use an RNA integrity score (RIS) or RNA integrity number (RIN) to assess RNA integrity.

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For best results, aim for an RIN value of ≥ 8. However, successful miRNA library prep is possible with samples whose RIN values are ≤ 8. For samples with low RIN values, increase the sequencing reads allocated per sample to allow for RNA degradation products. This allowance also applies to FFPE-derived RNA samples, which typically have low RIN values.

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It is not useful to assess the RNA integrity of total RNA derived from fluids and/or exosomes.

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Cell and tissue samples: The recommended starting amount of total RNA is 100 ng. You can use the protocol with 1–500 ng total RNA.

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Serum and plasma samples: The recommended starting amount of total RNA is 5 µl RNA eluate when 200 µl serum/plasma is processed using either of the following kits:

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QIAGEN miRNeasy Serum/Plasma Kit

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QIAGEN miRNeasy Serum/Plasma Advanced Kit

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Exosome samples prepared from serum and plasma: The recommended starting amount of total RNA is 5 µl RNA eluate when 1 ml of serum or plasma is processed using the QIAGEN exoRNeasy kits.