Dec 12, 2024
RNA Extraction and RT-PCR on Mouse tissues
- Jayshen Arudkumar1,2,
- Yu C.J. Chey1,2,
- Sandra Piltz1,2,
- Paul Quinton Thomas1,2,
- Fatwa Adikusuma1,2
- 1University of Adelaide;
- 2SAHMRI
Protocol Citation: Jayshen Arudkumar, Yu C.J. Chey, Sandra Piltz, Paul Quinton Thomas, Fatwa Adikusuma 2024. RNA Extraction and RT-PCR on Mouse tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3z3olk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 23, 2024
Last Modified: December 12, 2024
Protocol Integer ID: 94042
Keywords: RNA, RT-PCR, Dystrophin, transcript, Phenotyping, Knockout, DMD
Disclaimer
These protocols are for research purposes only.
Abstract
Paper abstract: CRISPR-Cas9 gene-editing technology has revolutionised the creation of precise and permanent modifications to DNA, enabling the generation of diverse animal models for investigating potential treatments. Here, we provide a protocol for the use of CRISPR-Cas9 to create murine models of Duchenne Muscular Dystrophy (DMD) along with a step-by-step guide for their phenotypic and molecular characterisation. The experimental procedures include CRISPR microinjection of embryos, molecular testing at the DNA, RNA, and protein levels, forelimb grip strength testing, immunostaining and serum creatine kinase (CK) testing. We further provide suggestions for analysis and interpretation of the generated data, as well as the limitations of our approach. These protocols are designed for researchers who intend on generating and using mouse models to study DMD as well as those seeking a detailed framework of phenotyping to contribute to the broader landscape of genetic disorder investigations.
Protocol summary: To obtain high-quality RNA for subsequent molecular analyses, specifically to assess mRNA transcript expression in mouse tissues. Following this, cDNA synthesis is employed to convert the isolated RNA into complementary DNA (cDNA). This cDNA serves as a stable template for downstream analyses, where we look at using reverse transcription PCR (RT-PCR) or quantitative real-time PCR (qPCR).
Image Attribution
BioRender was used to generate figures for this manuscript.
Materials
• TRIzolTM Reagent (Invitrogen).
• Chloroform.
• MagNA Lyser Green Beads (Roche)
• Ethanol.
• Isopropanol.
• DNase/RNase-free distilled water.
• RNEasy Mini Extraction kit (Qiagen)
• High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
• QuantStudio Real-Time PCR software v1.3 (Applied Biosystems)
Safety warnings
Wear proper PPE (gloves, safety goggles, enclosed shoes and lab coat) and prepare solvents in a chemical fume hood. Dispose used solvents or waste material in an appropriate biohazard waste containers.
Ethics statement
Animal work described in this manuscript has been approved and conducted under the oversight of the Animal Ethics Committee of South Australian Health and Medical Research Institute (SAHMRI) and The University of Adelaide.
Preparation Phase
Preparation Phase
Keep the tissue samples on ice
Set the centrifuge to cool at 4°C
Mince the heart and skeletal muscle tissues in a few drops of water and place the minced portions into a magNA lyser tube for bead homogenisation step
Homogenise samples in the Precellys bead-based homogeniser at 6500 RPM for 2 cycles at 20s each
TRIzol Treatment
TRIzol Treatment
Add 500 μL of Invitrogen TRIzol reagent to the supernatant
Incubate for 5 minutes at room temperature (RT) to dissociate the nucleoprotein complex
Chloroform Extraction
Chloroform Extraction
Add 100 μL chloroform
Note
Use 0.2 ml chloroform/ 1 ml TRIZOL reagent
Shake the tubes vigorously by hand for 15 seconds to mix
Incubate for 2 min at RT.
Centrifuge at 12,000 RCF in a 4°C cooled centrifuge for 15 minutes.
RNA Precipitation and Collection
RNA Precipitation and Collection
Label new microcentrifuge tubes and keep on ice
Transfer approximately 175 μL of the colourless liquid layer to a new eppendorf tube.
Add an equal volume of 70% ethanol. Mix by pipetting. Invert the tube and mix to disperse any visible precipitate that can form after adding ethanol.
Transfer a maximum of 700 μL to the QIAGEN spin column with a collection tube.
Centrifuge for 30 seconds at 8,000 RCF (room temperature centrifuge).
Place the flow-through back into the spin column and centrifuge again for 30 seconds at 6,500 RCF. Discard the flow-through and place the column back into the collection tube.
Add 700 μL Buffer RW1 to the RNeasy spin column. Centrifuge for 15 seconds at 8,000 RCF.
Add 500 μL Buffer RPE to the RNeasy spin column. Centrifuge for 15 seconds at 8,000 RCF.
Discard the flow-through and place the spin column in a new collection tube.
Add 500 μL Buffer RPE to the RNeasy spin column. Centrifuge for 2 minutes at 8,000 RCF to wash the spin column membrane.
Discard the flow-through and place the spin column in a new collection tube.
Place the RNeasy spin column in a new collection tube. Centrifuge for 1 minute at max speed.
Place the RNeasy spin column in a new QIAGEN RNase-free 1.5 ml tube.
Add 30-50 μL RNase-free water directly to the spin column membrane.
Centrifuge for 1 minute at 8,000 RCF to elute the RNA.
Note
For <100 μg starting tissue, use one 30-100 μL elution volume. For >100 μg, use 2–3 sequential 100 μL elutions.
Measure concentration of the RNA solution using NanoDrop Spectrophotometer
Note
The A260/A280 ratio should be approximately 2.0, but values between 1.8 and 2.1 are considered to be of acceptable purity.
Reverse Transcription Reaction for cDNA Generation
Reverse Transcription Reaction for cDNA Generation
Follow the protocol provided by the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) to convert 1-2 μg of RNA into cDNA
Dilute cDNA in 50 μL ultrapure water prior to running downstream analysis
Reverse-transcription polymerase chain reaction (RT-PCR)
Reverse-transcription polymerase chain reaction (RT-PCR)
The New England Biolabs (NEB) website can be consulted for the finer details of reaction volumes and components when amplifying using a standard NEB Taq DNA Polymerase and the 10X Standard Taq Reaction Buffer. The primers we used are listed in Supplementary Figure S2. We recommend using cDNA synthesised from 1-2 μg of RNA, as well as assembling all rection components on ice prior to transferring to the preheated thermocycler. After the RT-PCR, confirm the expected band sizes by carefully visualizing the amplified products on a 1% agarose gel.