High-quality RNA from cells isolated by laser capture microdissection.
Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.
Mikulowska-Mennis
A
A
Arcturus, Mountain View, CA 94043, USA. amennis@arctur.com
Taylor
T B
TB
Vishnu
P
P
Michie
S A
SA
Raja
R
R
Horner
N
N
Kunitake
S T
ST
D000818:Animals; D001921:Brain / Q000166:cytology; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D004210:Dissection / Q000295:instrumentation / Q000379:methods*; D005260:Female; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000166:cytology; D007668:Kidney / Q000166:cytology; D007834:Lasers; D008099:Liver / Q000166:cytology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011786:Quality Control; D012313:RNA / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012469:Salivary Glands / Q000166:cytology; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods; D013950:Thymus Gland / Q000166:cytology
D012313:RNA