Total RNA extraction from brain tissue with on-column DNA removal
Browse experimental protocolsExtract total RNA from brain tissue using TRIzol phase separation, a silica spin column and on-column DNase treatment.
Scope and method
Sample：Frozen brain tissue; record the tissue amount and sample identifier separately.
- Wistar ICV-STZ rat
- Age
- 3–4 months of age
- Sex
- Male
- Tissue
- Brain tissue
Bill of materials (BOM)
| Step | Material | Role | Amount | Required / optional | Substitution conditions | SKU / specification | Literature product |
|---|---|---|---|---|---|---|---|
| Clean dissection tools | Sharps container for razor-blade disposal | Collect used razor blades after brain tissue sample preparation. Used for sharps collection; the source does not report capacity, model or consumption per experiment. | Not reported in the source | Required | Not reported in source | Available for inquiry | Not reported in source |
| Clean dissection tools | Razor blades for tissue subdivision | Used to subdivide brain tissue samples during preparation; discard after use into a sharps container. The source uses razor blades but does not report their dimensions, material or quantity per sample. | Not reported in the source | Required | Not reported in source | Available for inquiry | Not reported in source |
| Prepare consumables | 1.5 ml microcentrifuge tubes for RNA extraction | Used for chloroform phase separation, aqueous-phase transfer and eluate collection during brain tissue RNA extraction. Volume 1.5 ml. The preparation step specifies three tubes per sample; additional replacement tubes in later steps must be accounted for separately. | 3 per sample during sample preparation; the total number of later replacement tubes is not reported in the source | Required | Not reported in source | Available for inquiry | Not reported in source |
| Prepare dry ice | Dry ice for brain tissue RNA extraction preparation | Used during brain tissue RNA extraction preparation to cool the tool container and tissue-sample container. The source specifies dry ice without reporting its mass or consumption per sample; two containers is not a dry-ice mass. | Not reported in the source | Required | Not reported in source | Available for inquiry | Not reported in source |
| Prepare consumables | Green-cap lysis tube containing white grinding beads | Use a separate lysis tube for each tissue sample for lysis and homogenization in TRIzol. The source describes a green cap and white grinding beads but does not report bead material, diameter or a manufacturer catalog number. | 1 per sample during sample preparation | Required | Not reported in source | Available for inquiry | Not reported in source |
| Load the column | PureLink RNA Mini Kit（12183018A） | RNA Column Purification Kit PureLink RNA Mini Kit, 12183018A; add ethanol to Wash Buffer II according to the instructions. | Load column 500 µl/time; Wash Buffer I 350 µl/time, Wash Buffer II 500 µl/time | Required | Not reported in source | Available for inquiry | Not reported in source |
| Prepare DNase mix | PureLink On-Column DNase Treatment Reagent | On-column DNA removal PureLink on-column DNase system; DNase reconstitution and buffers are prepared according to this system. | 80 µl DNase reaction mixture/sample | Required | Not reported in source | Available for inquiry | Not reported in source |
| Add ethanol | 70% ethanol for RNA purification | Ethanol for RNA column binding 70% ethanol, prepared with RNase-free water. | Equal volume with water, 400–600 µl | Required | Not reported in source | Available for inquiry | Not reported in source |
| Prepare chloroform tubes | chloroform | Lysate phase separation Chloroform; for use in the adopted TRIzol phase separation procedure. | 200 μl/sample | Required | Not reported in source | Available for inquiry | Not reported in source |
| Aliquot TRIzol | TRIzol Total RNA Extraction Reagent | Brain tissue lysis TRIzol Total RNA Extraction Reagent. | 1000 μl/sample | Required | Not reported in source | Available for inquiry | Not reported in source |
| Clean the workbench | 70% ethanol | Workbench and tool cleaning 70% ethanol; for cleaning only, not to be used as ethanol for RNA column purification. | According to the amount required to clean the surface | Required | Not reported in source | Available for inquiry | Not reported in source |
| Add elution water | Nuclease-free water | RNA elution RNase-free water. | 30 µl/sample | Required | Not reported in source | Available for inquiry | Not reported in source |
Inquire about the whole BOM
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Procedure
Check sample locations
Locate samples by the year, identifier, and well position of the sample box.
Record sample information
Record the sample identifier, animal identifier, genotype, age, and sex.
Record tissue amounts
Record the amount of tissue in each sample; very small samples do not need to be subdivided.
Clean the workbench
Spray the workbench with 70% ethanol or RNase Away.
Prepare dry ice
Prepare two containers of dry ice, one for dissection tools and one for tissue samples.
Clean dissection tools
Wipe the cutting board, blades, and forceps with 70% ethanol. Do not spray RNase Away onto tools in contact with dry ice because it will freeze; discard used blades in a sharps container.
Prepare consumables
For each sample, prepare and label one green-cap lysis tube containing white grinding beads, three 1.5 ml tubes, one spin column with collection tube, and one spare collection tube.
Reconstitute DNase
Reconstitute DNase with the supplied 550 µl RNase-free water.
Prepare DNase mix
Prepare 80 µl per sample: 8.0 µl 10X DNase I reaction buffer, 10.0 µl reconstituted DNase, and 62.0 µl RNase-free water.
Retrieve lysis reagent
Remove TRIzol from the 4°C refrigerator and place it in a fume hood.
Aliquot TRIzol
In a fume hood, add 1000 μl TRIzol to each lysis tube.
Add brain tissue
Subdivide samples according to tissue amount, place them in their respective lysis tubes, and mix by inversion.
Submerge the tissue
Completely submerge the tissue in TRIzol, using a separate lysis tube for each sample.
Clean the tools
Wipe tools with 70% ethanol before contacting samples, between samples, and after use.
Storage during a pause
If a pause is needed, samples submerged in TRIzol can be stored at -80°C before homogenization.
Prepare cooling
If using wet-ice cooling rather than working in a cold room, replace the dry ice in the tool container with wet ice.
Homogenization
Process each sample with a tissue homogenizer.
Select the homogenization program
Select the recommended program for the tissue type; most programs run for 40 seconds.
Check homogenization
Homogenize until the tissue is dispersed as fine particles in TRIzol. Small brain tissue pieces usually require one 40 second homogenization round; larger organs or larger tissue amounts may require three rounds. Pink foam after brain tissue homogenization usually dissipates within a few minutes.
Cool between homogenization rounds
If repeated homogenization is needed, place the lysis tube on wet ice or in a cold room for 2-mins between rounds to avoid RNA degradation caused by heat from homogenization.
Incubate the lysate
Allow the TRIzol homogenate to stand for 5 mins.
Prepare chloroform tubes
In a fume hood, add 200 μl chloroform to a new 1.5 ml tube.
Transfer the lysate
In a fume hood, add 1000 μl TRIzol lysate to the tube containing chloroform. Do not add the 200 μl chloroform directly to the lysis tube containing grinding beads because this may interfere with phase separation.
Mix the phase-separation solution
Mix gently by inversion for 15-30 secs.
Incubate at room temperature
Allow to stand at room temperature for 2-3 mins.
Low-temperature phase-separation centrifugation
Centrifuge at 4°C and 12,000xg for 15 mins.
Transfer the aqueous phase
Transfer 400 μl of the upper aqueous phase to a new 1.5 ml tube; a P200 may be used for two transfers of 200 μl each.
Add ethanol
Add 400-600 μl of 70% ethanol to the RNA-containing aqueous phase.
Mix the column-loading solution
Pipette gently 4-5 times, maintaining a 1:1 ratio of aqueous phase to ethanol, until no RNA floccules remain visible. Working aliquots of 70% ethanol may be kept at room temperature in 15 ml tubes. Prepare 70% ethanol from 70 ml absolute (100%) ethanol and 30 ml RNase-free water; store the stock at 4°C.
Load the column
Load 500 µl of sample onto a spin column with a collection tube.
Centrifuge for binding
Centrifuge at room temperature and 12,000xg for 20 secs.
Discard the flow-through
Discard only the flow-through and retain the collection tube.
Process the remaining sample
Repeat loading, centrifugation, and flow-through disposal until all sample has passed through the column membrane. RNA remains on the membrane and is subsequently eluted with RNase-free water.
On-column DNase treatment
Add 350 µl Wash Buffer I to the spin column and centrifuge at room temperature and 12,000g for 20 secs. Discard the flow-through and collection tube and replace the collection tube. Add 80 µl PureLink DNase mix to the center of the membrane and incubate at room temperature for 15 mins. Add another 350 µl Wash Buffer I, centrifuge at room temperature and 12,000g for 20 secs, discard the flow-through and collection tube, replace the collection tube, and continue with Wash Buffer II washing.
Add wash buffer
Add 500 µl Wash Buffer II to the spin column.
Wash centrifugation
Centrifuge at room temperature and 12,000xg for 20 secs.
Discard the wash flow-through
Discard only the flow-through and retain the collection tube.
Repeat washing
Repeat the addition of Wash Buffer II, centrifugation, and disposal of the flow-through once more.
Centrifuge to dry
Centrifuge at room temperature and 12,000xg for 1 min to dry the membrane.
Replace the elution tube
Discard the flow-through and collection tube and place the column in a new 1.5 ml tube.
Add elution water
Add 30 µl RNase-free water to the center of the membrane.
Incubate for elution
Allow to stand at room temperature for 30 secs.
Collect RNA
Centrifuge at room temperature and 12,000xg for 2 mins; for DNase-treated samples, reduce centrifugation to 1 min. If the collection tube is damaged, transfer the eluate to a new 1.5 ml tube.
Store RNA
Store RNA at -80°C; keep it on wet ice during handling and avoid prolonged exposure to room temperature.
Quality control and readouts
Record tissue amount, RNA concentration, and integrity for each sample. Avoid RNase contamination and residual ethanol. Include no-reverse-transcription controls in downstream gene expression assays to assess residual DNA.
DNase-treated total RNA, together with RNA concentration and quality records.
References
- Bernlohr DA, Martell Martinez H. Brain Tissue RNA Extraction -- University of Minnesota TMCs. protocols.io v1, 2024-06-17. · DOI: 10.17504/protocols.io.bp2l62dmdgqe/v1
- Thermo Fisher Scientific. PureLink RNA Mini Kit User Guide, MAN0000406, 14 June 2012, pp.11,50–53,63.
- Schisandrin ameliorates cognitive deficits, endoplasmic reticulum stress and neuroinflammation in streptozotocin (STZ)-induced Alzheimer’s disease rats. PMID:32336744. · DOI: 10.1538/expanim.19-0146