Having difficulties with your experiment?
We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios with your laser capture microdissection (LCM) experiments.
View the relevant questions below:
The dissection and embedding steps are critical for the RNA quality for LCM samples. We suggest using the entire adjacent section or dissecting a large area of the section (>1000 cells) for RNA isolation. Before proceeding to LCM microdissection, we recommend assessing the quality of RNA isolated from the adjacent section. With good amount of RNA, one can assess the RNA quantity and quality using the Qubit™ RNA HS Assay Kit and the Agilent Bioanalyzer. This will help to make sure that good quality RNA is obtained from a low number of LCM cells.
Formalin-fixed paraffin-embedded (FFPE) samples introduce unique challenges for gene expression profiling and gene expression quantitation, including chemical modification and fragmentation of RNA molecules. The initial paraffin blot preparation and storage significant affect the RNA quality. Archival FFPE samples present additional challenges due to increased RNA degradation over time. As a result, not all FFPE samples contain high quality RNA.
In addition, a typical Bioanalyzer profile of RNA from an FFPE sample can look fragmented and degraded and as a result, that approach may not be useful in determining the transcribability of your RNA. Therefore, a tissue scrape test is recommended. You can use the entire adjacent section to isolate RNA and to access the RNA quantity and quality for intact RNA. Two sets of β-actin gene-specific primers designed to amplify short segments of DNA within the β-actin target sequence from the 5’ and 3’ of the β-actin gene can be used to estimate the amount of RNA in a given sample in relation to β-actin content using real-time PCR. The RNA quantity derived from the 3' primer set is used to quantitate the RNA in the FFPE tissue scrape. The ratio of the RNA yield obtained from both sets of PCR primers is the 3'⁄5' ratio and is used as an indication of RNA quality. If there is less product yield of the 5’ β-actin primers, it indicates that there is less full length of RNA.
Here are possible causes and solutions:
- The sample may contain residual water. Ensure that the ethanol solution is fresh. Ethanol is hygroscopic. Keep the ethanol bottle tightly capped, and do not pour ethanol solution until you are ready to use it. If you suspect that the 100% ethanol solution has absorbed water, purchase a new bottle.
- The sample may have dried between protocol steps. Carry out the “Staining and Dehydration” segment of the protocol at a steady pace.
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