Journal: Frontiers in Molecular Neuroscience
Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq
Figure Lengend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
Article Snippet: In addition, we also tested whether different methods of RNA isolation had an impact on RNA quality and found that kits with minimal digestion time and temperature, such as 5 min at room temperature in the QIAGEN Micro RNeasy kit, resulted in superior quality RNA (RNA Integrity Number, RIN, 9–10, 10 being intact RNA) compared to longer digestion times at higher temperature, such as 30 min at 42°C in the Arcturus PicoPure RNA Isolation Kit (RIN 6–7), which is the current standard kit used in studies using LCM.
Techniques: Laser Capture Microdissection, Isolation