title: Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.
abstract: Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6-7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.
authors.affiliation: Larry L. Hillblom Islet Research Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Butler
authors.affiliation: Larry L. Hillblom Islet Research Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA ; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Matveyenko
authors.affiliation: Larry L. Hillblom Islet Research Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Kirakossian
authors.affiliation: Larry L. Hillblom Islet Research Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Johanna
authors.initials: J
authors.lastname: Park
authors.affiliation: Larry L. Hillblom Islet Research Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Gurlo
authors.affiliation: Larry L. Hillblom Islet Research Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA ; Jonsson Comprehensive Cancer Center, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Butler
keywords: Islets; Laser capture microdissection – LCM; PDGs; Pancreas; RNase inhibitors