TY - JOUR
T1 - Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas
AU - Butler, Alexandra E.
AU - Matveyenko, Aleksey V.
AU - Kirakossian, David
AU - Park, Johanna
AU - Gurlo, Tatyana
AU - Butler, Peter C.
N1 - Publisher Copyright:
© 2016, © National Society for Histotechnology 2016.
PY - 2016/4/2
Y1 - 2016/4/2
N2 - 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.
AB - 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.
KW - Islets
KW - Laser capture microdissection – LCM
KW - PDGs
KW - Pancreas
KW - RNase inhibitors
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U2 - 10.1080/01478885.2015.1106073
DO - 10.1080/01478885.2015.1106073
M3 - Article
AN - SCOPUS:84978514990
SN - 0147-8885
VL - 39
SP - 59
EP - 65
JO - Journal of Histotechnology
JF - Journal of Histotechnology
IS - 2
ER -