RNA isolation methods

Different RNA extraction methods

1. RNA - ISOLATION

2. RNA extraction  RNA extraction is the purification of RN A from biological samples. This procedure is complicated by the presence of ribonuclease enzymes in cells and tissues.

3. Isolation of RNA Requires STRICT precautions to avoid sample degradation. RNA especially labile.

4. RNAses RNases are naturally occurring enzymes that degrade RNA Common laboratory contaminant (from bacterial and human sources) Also released from cellular compartments during isolation of RNA from biological samples Can be difficult to inactivate

5. RNAses RNAses are enzymes which are small proteins that can renature and become active. MUST be eliminated or inactivated BEFORE isolation. CRITICAL to have a separate RNAse free area of lab.

6. Protecting Against RNAse Wear gloves at all times Use RNase-free tubes and pipet tips Use dedicated, RNase-free, chemicals Pre-treat materials with extended heat (180 C for several hours), wash with DEPC-treated water, NaOH or H2O2 Supplement reactions with RNase inhibitors

7. TRIZOL RNA Isolation Protocol TRIZOLE REAGENT The correct name of the method is guanidinium thiocyanate- phenol-chloroform extraction . TRIzol is light sensitive and is often stored in a dark-colored, glass container covered in foil. It must be kept below room temperature. When used, it resembles cough syrup, bright pink. The smell of the phenol is extremely strong.. Caution should be taken while using TRIzol (due to the phenol and chloroform ). Exposure to TRIzol can be a serious health hazard. Exposure can lead to serious chemical burns and permanent scarring . A lab coat, gloves and a plastic apron are recommended

8. PRINCIPLE TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues. TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. RNA remains only in the aqueous phase. After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. But the DNA and proteins can recover by sequential separation after the removal of aqueous phase. Precipitation with ethanol requires DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA. This RNA can be used in Northern blot analysis, rt- pcr , in vitro translation, RNase protection assay, and molecular cloning

9. Required reagents: DEPC-treated water TRIzol Reagent Ice cold PBS Cell scraper 70% ethanol Isopropyl alcohol B. Equipment and supplies: Refrigerated centrifuge Microcentrifuge Micropipettors Aerosol-barrier tips Vortex mixer Powder-free gloves Centrifuge tubes

10. Homogenization: Tissues: Homogenize tissue samples in 1 ml of TRIZOL reagent per 50 to 100 mg of tissue using a glass-Teflon or power homogenizer. Cells grown in Monolayer: Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of TRIZOL Reagent per 3.5 cm diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly.

11. 2. Phase Separation    The homogenized samples were incubated for 5 minutes at 15 to 30°C for the complete dissociation of nucleoprotein complexes. 0.2 ml (200 microliters)of chloroform per 0.75 ml of TRIZOL LS Reagent was added. The tubes were shaked vigorously by hand for 15 seconds and incubated them at 15 to 30°C for 2 minutes. The samples were centrifuged for 15 minutes at no more than 12,000 g (4°C). The aqueous phase was transferred to other tubes. ( Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains only in the aqueous phase. The volume of the aqueous phase is about 70% of the volume of TRIZOL Reagent used for homogenization.)

12. Phase Separation 

13. 3. RNA Precipitation   The RNA was precipitated from the aqueous phase by mixing with 3 microlitre of glycogen and 500 microlitre of isopropyl alcohol. The mixture was centrifuged for 30 minutes at 12,000 × g (2 to 8°C).( The RNA precipitate forms a gel-like pellet on the side of the tube at bottom).

14. 4. RNA Wash   The supernatant was removed. The RNA pellet was washed once with 75% ethanol, adding 900 microlitre of 75% ethanol per 0.75 ml of TRIZOL Reagent used for the initial homogenization. The sample were inverted and mixed and centrifuged at 12,000 rpm for 30 minutes at 4 degree.

15. 5. Redissolving RNA   The RNA pellet was dried . RNA was dissolved in RNase-free water (or 0.5% SDS solution) by passing the solution through the pipette tip for a few times, and incubating for 10 minutes at 55 to 60°C.    

16. 6. SPECTROPHOTOMETRIC ANALYSIS: Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40 dilution). Using 10 μl microcuvette, take OD at 260 nm and 280 nm to determine sample concentration and purity. The A260/A280 ratio should be above 1.6. Apply the convention that 1 OD at 260 equals 40 µg /ml RNA. Example: Use the buffer in which the RNA is diluted to zero the spectrophotometer: Volume of RNA sample = 100 µl Dilution = 10 µl of RNA sample + 390 µl distilled water (1/40 dilution) Absorbance of diluted sample measured in a cuvette (RNase-free): A260 = 0.23 Concentration of original RNA sample = 40 x A260 x dilution factor = 40 x 0.23 x 40 RNA concentration: 368 µg/ml 0r 368 ng/ul

17. Other Methods of RNA ISOLATION Filter-based RNA isolation Magnetic Particle Methods

18. Filter-based RNA isolation Filter-based, spin basket formats utilize membranes that are seated at the bottom of a small plastic basket. Samples are lysed in a buffer that contains RNase inhibitors (usually guanidine salts),are bound to the membrane by passing the lysate through the membrane using centrifugal force. Wash solutions are passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation. 

19. Filter-based RNA isolation

20. Filter-based RNA isolation Benefits of spin basket formats Convenience and ease of use Ability to isolate RNA and DNA. Ability to manufacture membranes of various dimensions Drawbacks of spin basket formats Propensity to clog with particulate material Retention of large nucleic acids such as gDNA

21. Magnetic Particle Methods Magnetic particle methods utilize small (0.5–1 µm) particles that contain a paramagnetic core. Paramagnetic particles migrate when exposed to a magnetic field, but retain minimal magnetic memory once the field is removed. This allows the particles to interact with molecules of interest based on their surface modifications, be collected rapidly using an external magnetic field, and then be resuspended easily once the field is removed. Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic particles. The magnetic particles and associated cargo are collected by applying a magnetic field.

22. Magnetic Particle Methods

23. Magnetic Particle Methods

24. Videos https://www.youtube.com/watch?v=RmPsLoIPRwc&ab_channel=Abnova https://www.youtube.com/watch?v=Kiu1tZVlvIw&ab_channel=CDRT-tube

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