Quantification of Estrogen Receptor Messenger RNA by Quantitative Polymerase Chain Reaction Using Internal Standard Fragment
| Autor: | Sumitaka Miyamoto, Hideki Mizunuma, Tsutomu Uchida, Yoshito Ibuki, Issay Kagami |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Male
DNA Complementary Transcription Genetic Biophysics Estrogen receptor Biology Polymerase Chain Reaction Biochemistry law.invention Endometrium chemistry.chemical_compound law Complementary DNA Animals Humans RNA Messenger Rats Wistar Molecular Biology Polymerase chain reaction DNA Primers Messenger RNA Base Sequence Estradiol Fragment (computer graphics) Uterus Reproducibility of Results RNA Cell Biology Reference Standards Molecular biology Recombinant Proteins Endometrial Neoplasms Rats Mutagenesis Insertional Electrophoresis Real-time polymerase chain reaction Receptors Estrogen chemistry Agarose Female Orchiectomy |
| Zdroj: | Biochemical and Biophysical Research Communications. 228:358-364 |
| ISSN: | 0006-291X |
| DOI: | 10.1006/bbrc.1996.1666 |
| Popis: | A simple and reliable polymerase chain reaction-based method for quantifying human and rat estrogen receptor (ER)-mRNA is described. This method involves co-amplification of cDNA transcribed from sample RNA with an internal standard to reduce tube to tube amplification variations. Human and rat internal standards were synthesized by insertion of a DNA fragment near the midportion of human and rat target ER cDNA molecules. After co-amplification, both products of target ER cDNA and internal standard were separated on agarose gel by electrophoresis and transferred onto a membrane filter. The radioactivity hybridized with32P-labeled ER cDNA was counted. The ER mRNA content was calculated by linear regression analysis after obtaining a logarithm of ratios between the sample and the internal standard radioactivity. The coefficient of variation of this assay was 18.8%. An absolute amount of ER mRNA in less than 104cultured cells could be measured. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect