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RNA quantitative analysis from fixed and paraffin-embedded tissues: Membrane hybridization and capillary electrophoresis
- Source :
- Scopus-Elsevier
-
Abstract
- Fixed and paraffin-embedded tissues from pathology department archives are available for RNA expression analysis. We describe a general method for quantitation of specific RNA sequence extracted from single 6–8-μm human histological tissue sections cut from paraffin blocks. For each specific mRNA, the range of linear relationship between the log of the initial total RNA concentration and the log of the specific product after reverse transcription (RT)-PCR must be established. We usually perform RT with avian myeloblastosis virus (AMV)-RT, using specific antisense primers and a variable number of cycles of PCR amplification. The number of cycles must be adjusted within the range in which a linear relationship exists between the log of the amount of amplification product and the number of cycles. The quantity of specific product is standardized relative to β-actin mRNA to normalize for the degree of RNA degradation, which can be quite different among samples. The amplification products were quantified by dot blot and 32P-labeled hybridization probe or by capillary electrophoresis with a laser-induced fluorescence detector. The intratest variation range was for the dot blot mean ±10% standard deviation (SD) and for the capillary electrophoresis mean ±3% SD.
- Subjects :
- Tissue Fixation
Receptor, ErbB-2
Aves, Avian myeloblastosis virus
Messenger
Gene Expression
Dot blot
Polymerase Chain Reaction
Capillary
cell hybridization
Neoplasms
Antisense, Electrophoresi
erbB-2, RNA
Gel electrophoresis
Paraffin Embedding
Hybridization probe
Epidermal Growth Factor, Receptor
article
Nucleic Acid Hybridization
ErbB Receptors
Oligodeoxyribonucleotides
Capillary, Fluorescence, Gene Expression, Humans, Neoplasms, Nucleic Acid Hybridization, Oligodeoxyribonucleotides, Paraffin Embedding, Phosphorus Radioisotopes, Polymerase Chain Reaction, Receptor
Aves
Receptor
Biotechnology
Electrophoresis
rna analysis
capillary electrophoresis
Biology
DNA, Antisense
Fluorescence
General Biochemistry, Genetics and Molecular Biology
Nucleic acid thermodynamics
Capillary electrophoresis
Humans
human
RNA, Messenger
Antisense
erbB-2
rna sequence
Messenger RNA
Epidermal Growth Factor
Avian myeloblastosis virus
Electrophoresis, Capillary
RNA
DNA
Actins, DNA
Molecular biology
human tissue
Actins
Reverse transcriptase
Messenger, Tissue Fixation
Phosphorus Radioisotopes
article, capillary electrophoresis, cell hybridization, human, human tissue, rna analysis, rna sequence
Antisense, Electrophoresis
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Scopus-Elsevier
- Accession number :
- edsair.doi.dedup.....92a926f4a567dc404421f72077ff49a3