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RNA Extraction from Wistar Rat Cochlea for qRT-PCR
Published: Dec 5, 2017 DOI: 10.21769/BioProtoc.2621 Views: 7382
Edited by: Gal Haimovich
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Abstract
Otology research has developed considerably in recent years and molecular analysis is crucial to identify metabolic pathways and therapeutic targets. However, the structure of the cochlea limits the amount of cell mass, and special care is required for RNA extraction. Studies applying this technique to the cochlea are scarce in the literature, and difficulties in this area exist. In the context, we describe a method for extracting RNA in Wistar rats for qRT-PCR analyses in order to clarify certain aspects and obtain good quality material.
Keywords: Cochlea/metabolism
RNA/chemistry
Real-time polymerase chain reaction
Background
Understanding the molecular mechanisms involved in damage to hair cells is important to preserve hearing. qRT-PCR from mRNA is used in the study of metabolic pathways and possible therapeutic targets. Otological studies typically use small rodents, although the isolation of sensory cell samples with well-preserved RNA for transcriptional analysis is a significant challenge. Studies conducted with these animals have presented efficient methods for extracting RNA in the neonatal cochlea, however, adult cochlea presents limitations, due to the recovery of small amounts of mRNA. To evaluate the quality of the obtained RNA, ultraviolet absorbance and agarose gel electrophoresis can be performed. Herein, we describe a method to obtain mRNA from adult Wistar rats that can be subsequently used in qRT-PCR assays.
Materials and Reagents
- Pipette tips (Eppendorf, catalog number: 0030014600 )
- Safe seal 2 ml microtubes (SARSTEDT, catalog number: 72.695.500 )
- RNase-free microcentrifuge tubes (Sigma-Aldrich, catalog number: Z717533 )
- Male Wistar rats obtained from the University of São Paulo
Note: Eighteen male Wistar rats were used as subjects, weighing on average 50-200 g, housed under controlled conditions (temperature 22-23 °C and humidity 60 ± 5%), a 12 h light/dark cycle and food/water ad libitum. Animal handling and well-being conformed to current national Brazilian regulations regarding protection and care of animals used for scientific purposes. All procedures were approved by the Committee on Ethics of Animal Experimentation of Medical school of University of São Paulo and approved under protocol 146/15. To obtain an adequate amount of mRNA two rats, four cochleae are needed. - RNaseZap® solution (Sigma-Aldrich, catalog number: R2020 )
- Isoflurane (Sigma-Aldrich, CAS number: 26675-46-7 )
- RNAlater® RNA stabilization solution (QIAGEN, catalog number: 76106 )
- TRIzol® (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596018 )
- Chloroform (Sigma-Aldrich, catalog number: C2432 )
- Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510 )
- MillenniumTM markers (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM7150 )
- Pure Link® RNA Mini Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12183018A )
- Pure Link® DNase for on-column (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12185010 )
- RNase free water from the Pure Link® RNA Mini Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12183018A )
- Agarose (Sigma-Aldrich, catalog number: A9539 )
- Pure Ethyl alcohol (Sigma- Aldrich, CAS number: 64-17-5 )
- Tris (1 M, pH 7.0) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9851 )
- Ethyl acetate (Sigma-Aldrich, CAS number: 141-78-6 )
- Ethylenediaminetetraacetate acid (EDTA) (Sigma-Aldrich, CAS number: 60-00-4 )
- 70% ethanol (see Recipes)
- 1% TAE agarose gel (see Recipes)
- 50x stock of TAE (see Recipes)
Equipment
- Pipettes 2-20 µl, 20-200 µl, 100-1,000 µl Eppendorf Reference® 2, pack triplo (Eppendorf, catalog number: 4920000911 )
- Forceps: Wagner scissors (Fine Science Tool, catalog number: 14068-12 ); Mayo-scissors (Fine Science Tool, catalog number: 14012-15 ); Dumont #5 forceps (Fine Science Tool, catalog number: 11251-10 )
- Rotor-stator (IKA, model: T10 basic , catalog number: 6924-71)
- Cold centrifuge (Eppendorf, model: 5427 R , catalog number: 5409000012)
- Room temperature centrifuge (Eppendorf, model: MiniSpin® plus , catalog number: 5452000018)
- Vortex (VWR® Fixed Speed Vortex Mixer) (VWR, catalog number: 10153-834 )
- NanoDropTM 2000/2000c spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000/2000c , catalog number: ND-2000)
- RT-PCR Thermal cycle–Step one (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM , catalog number: 4376598)
Procedure
Category
Molecular Biology > RNA > qRT-PCR
Cell Biology > Tissue analysis > Tissue isolation
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