Genomics Application Notes

MicroRNA Extraction from Laser Capture Micro- Dissected (LCM) FFPE Tissue Using Beckman Coulter's Agencourt FormaPure Kits

Summary Obtaining specific populations of cells from heterogeneous tissue samples is a major challenge in studying gene expression profiles of specific cell types. However, laser capture micro-dissection (LCM) enables the selection and capture of individual cells or groups of cells from discrete morphological structures by cutting away unwanted cells. This enriches histologically pure cell populations and therefore it can specifically isolate cancer cells from normal tissues. MicroRNA (miRNA), RNA, DNA or proteins can then be extracted from these micro-sections for gene expression analysis. LCM sampling can be applied to different samples, including formalin-fixed, paraffin-embedded (FFPE) tissues or fresh frozen tissues (FFT). This application note describes the purification of miRNA and total RNA from LCM FFPE samples using the Beckman Coulter Agencourt FormaPure kit. The method uses Beckman Coulter’s SPRI (Solid Phase Reverse Immobilization) magnetic bead-based chemistry, which provides an easy, rapid, high yielding, robust and automation-friendly nucleic acid purification procedure, that does not require vortexing, centrifugation or filtration steps. The data shows that miRNA was successfully extracted from LCM FFPE samples. Materials and Methods Tumor and normal cells from a prostate cancer FFPE tissue block were selected and micro-dissected using a digital pathology-guided approach to identify the regions of interest (ROIs). Three representatives matching LCM scanned sections were selected for study using the ArcturusXT ™ LCM Instrument (Life Technologies) and Arcturus ® Paradise ® FFPE Tissue LCM Staining Kit (Life Technologies, KIT03 1 2-J). The micro-dissected sections were then digested with lysis buffer and

proteinase K overnight using a FormaPure Kit (Beckman Coulter, A33342). Six samples (triplicates of tumor and benign samples) were used for miRNA extraction following the FormaPure Kit miRNA supplemental protocol (Beckman Coulter, AAG-666SP 11 . 1 4-A). Samples were eluted in 20 μL of nuclease-free water in the final elution step. The concentration of the RNA was measured by a Quant-iT ™ RiboGreen ® RNA assay kit (Life Technologies, R 11 490). One μL of RNA was analyzed by an Agilent RNA 6000 Pico chip and a small RNA chip (Agilent Technologies, 5067- 1 5 1 3 and 5067- 1 548) using the 2 1 00 Bioanalyzer (Agilent Technologies). miRNA (miR 1 46a and RNU48) gene expression was determined by a TaqMan ® microRNA assay (Life Technologies, assay ID00 1 097 and ID00 1 006). 1 0 ng of RNA was used for the reverse transcription reaction using the TaqMan ® microRNA Reverse Transcription Kit (Life Technologies, 4366596), and 1 .33 μL of cDNA was used per PCR reaction in triplicate using TaqMan ® Universal Master Mix II (Life Technologies, 4440038). Results and Discussion RNA Yields From LCM FFPE Samples Using FormaPure Kits Three pairs of tumor/benign LCM samples prepared from the FFPE block were used to extract total RNA and miRNA. The RNA was eluted in 20 μL of nuclease-free water. The RNA concentrations were between 2 ng/μL to 3.6 ng/μL (data not shown). The

AAG-621APP10.14-A