The spike-in ratios should be chosen in concordance with the starting input amount of total RNA, the desired final SIRV content, and the RNA content of the sample. In addition SIRV SpikSpike-In Calculation
Worksheets for each SIRV-Set are available via: https://www.lexogen.com/sirvs/download/.
The equations for calculating the SIRV spike in amounts are also explained in more detail in the SIRV User Guides (https://www.lexogen.com/docs/sirvs-mixes/).
Typically, for high quality RNA samples, we recommend to aim aiming for 1% of the sequencing reads mapping to the SIRV reference annotation (SIRVome). Please note that the target RNA content will vary between samples. If you do not know the target RNA content of your sample , you can assume a measure of 2 – 3%, which corresponds to the approximate RNA 3% for mRNA-Seq Library Preps and 4% for rRNA-depletion / total RNA-Seq Library Preps. As an example, the approximate mRNA content of reference RNA samples such as the Universal Human Reference RNA (UHRR) is approximately 2-3%.
For input RNA of low complexity or low quality, including from FFPE tissues, the RNA content is often lower or highly variable. SIRV-mapped reads will be at a higher fraction in these samples due to the intact nature of the SIRVs. Here there are two approaches:
Assume an RNA content of 3% and target a lower fraction of reads mapping to SIRVs, e.g., between 0.1 – 0.5%
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Assume a lower RNA content of 0.2 – 1% and
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target 1% of reads mapping to SIRVs.
Altering these assumptions will affect the eventual amount of SIRV RNA to spike into each sample, according to the starting input amount.
Similarly, RNA samples that are enriched for subsets of particular RNAs or cellular fractions can also have variable RNA content. For assistance with estimating the optimal spike-in percentage, or RNA content of your samples please contact support@lexogen.com.
How can I use SIRVs to measure the mRNA content of my sample?
For samples with unknown mRNA content we recommend to assume a 3% mRNA content, and spike in SIRVs to target 1% of final mapped reads. By comparing the share of reads aligning to the reference genome and the "SIRVome", respectively you can then derive the mRNA content. For further details, please consult the SIRV User Guides (https://www.lexogen.com/docs/sirvs-mixes/).