Use of laser capture microdissection for analysis of retinal mRNA/miRNA expression and DNA methylation.
Laser capture microdissection (LCM) is a useful method to isolate specific cells or cell layers of interest from heterogeneous tissues, such as the retina. The collected cells can be used for DNA, RNA, or protein analysis. We have applied LCM technology to isolate cells from the outer nuclear, inner nuclear, and ganglion cell layers of the retina for mRNA and microRNA (miRNA) expression and epigenetic (DNA methylation) analysis. Here, we describe the methods we have employed for sample preparation, LCM-based isolation of retinal layers, RNA/DNA extraction, RNA quality check, microRNA analysis by quantitative PCR, and DNA methylation analysis by bisulfite sequencing.
Avidin Ltd, Szeged, Hungary.
Laszlo
L
Hackler
Tomohiro
T
Masuda
Verity F
VF
Oliver
Shannath L
SL
Merbs
Donald J
DJ
Zack
D000818:Animals; D019175:DNA Methylation; D018076:DNA, Complementary / Q000138:chemical synthesis; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D061346:Laser Capture Microdissection / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D011786:Quality Control; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012160:Retina / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D035683:MicroRNAs; D012333:RNA, Messenger; D012313:RNA