Deep sequencing of entire population of expressed miRNAs in a given sample simultaneously queries thousands of miRNA sequences with extraordinary sensitivity and ability to detect novel ones. In general, the approach is used to give insights in how post-transcriptional regulation contribute to phenotype and for the discovery and development of novel biomarkers for diagnostic testing. In addition, given its extreme sensitivity, miRNA-seq is suitable for detection of both RNA and DNA viruses, as well as viroids that can be missed using targeted detection methods.
Here is the latest update of our improved miRNA-seq workflow for inputs below 100 ng of total RNA. We implemented QIAseq protocol indicated for samples with ultralow input levels (as little as 1 ng of total RNA!) to prepare libraries from RNA extracted from several challenging substrates: grapevine branch tissue, goat milk exosomes and bovine serum. In addition, we also included a sample with very low total RNA quantity originating from mouse fresh frozen tissue.
As shown in the figure below, despite of the sample scarcity we were able to obtain a typical miRNA-sized library peak at expected ~180 bp.
Figure. High Sensitivity DNA Assay 2100 Bioanalyzer traces of miRNA-sized library.
These results are really encouraging for all of those working with the limited samples such as biofluids (serum, plasma, CSF, urine) and FFPE tissues. An additional advantage of this particular procedure is that the kit integrates Unique Molecular Indices (UMIs) during the reverse-transcription process, ensuring that during data analysis all PCR and sequencing bias are eliminated.
Hope you found this post useful.
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