The quality of RNA is of great importance in gene expression studies. It is mostly measured using the RNA integrity number (RIN). Lately it has been shown that samples with low RIN and different fragmentation patterns could affect quality of sequencing data. For such low RIN samples a new approach has been developed by Illumina called the DV200 metric, which is the percentage of fragments >200 nucleotides. For samples with low RIN, DV200 has proved to be a better method to predict if good quality data from RNA sequencing can be generated. However, neither RIN nor DV200 provide spatial infromation on the RNA integrity. Thus, tissues with areas of heterogeneous RNA integrity, where regions of good quality RNA sequencing data could be generated from are missed. We have designed a method to spatially evaluate the RNA integrity in tissue, which we named in situ RNA QC. The method uses three probes with three different fluorophores, each bound to three specific cDNA regions synthesized from the high abundant and well conserved 18S rRNA. With the help of in-house technology from the Spatial Transcriptomics (ST) group at SciLifeLab, we enable creation of heat maps over the RNA integrity to show spatial fragmentation patterns of RNA in tissue. This could reveal the regional quality of transcripts in situ, which is crucial knowledge when selecting samples for further RNA sequencing. The assay has been tried using different tissue fixation methods in order to show a proof of concept that formalin gives shorter cDNA fragments than acetone. The generated heat-map provides a visual overview of RNA integrity in situ; hence this method could be used to select samples for sequencing by evaluation the spatial quality of RNA. For instance from fresh frozen and formalin fixated paraffin embedded (FFPE) tissue (biobanks contain large number of longterm storage FFPE samples). With this assay we will be able to determine which samples are suitable for sequencing.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-176873 |
| Date | January 2015 |
| Creators | Carlberg, Konstantin |
| Publisher | KTH, Skolan för bioteknologi (BIO) |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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