High-quality RNA from cells isolated by laser capture microdissection
| Autor: | S A Michie, T B Taylor, P Vishnu, S T Kunitake, N Horner, A Mikulowska-Mennis, R Raja |
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| Rok vydání: | 2002 |
| Předmět: |
Male
Quality Control Cell Separation Thymus Gland Biology In Vitro Techniques Kidney General Biochemistry Genetics and Molecular Biology Salivary Glands Mice Complementary DNA Intestine Small Animals Gene Microdissection Laser capture microdissection Mice Inbred BALB C Staining and Labeling Reverse Transcriptase Polymerase Chain Reaction Dissection Lasers RNA Brain Ribosomal RNA Molecular biology Staining Liver Female RNA extraction Biotechnology |
| Zdroj: | BioTechniques. 33(1) |
| ISSN: | 0736-6205 |
| Popis: | Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNA from 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 Kit for 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. |
| Databáze: | OpenAIRE |
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