Lab 2: RNA Isolation

Lab Objectives

• Complete RNA isolation procedure
• Design primers
• Set up group experiments and prepare detailed schedule for maintaining experiments

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IMPORTANT SAFETY NOTES

Wear clean gloves! For your own safety as well as the integrity of your RNA samples, you must wear gloves throughout this week's lab. Phenol and chloroform are nasty, cuastic chemicals, so gloves are necessary when handling anything that comes in contact with either reagent. Additionally, RNases are constantly secreted from your skin and can easily enter, and subsequently degrade, your RNA sample.

Phenol/Chloroform Handling and Disposal

• Handling - You must wear gloves, safety glasses and lab coats at all times! These chemicals have potential to do damage to clothing and exposed body parts. TriReagent may be used on the benchtop, but be aware that it is caustic, very volatile and has a very strong odor. Chloroform must only be used in a fume hood! It is extremely caustic, volatile, and inhalation of fumes can be dangerous.
• Disposal - All tips/tubes/gloves that come in contact with phenol/chloroform must be disposed of in the "Solid Phenol/Chloroform Waste" container found in the fume hood. None of this type of waste should be discarded in regular trash! Any liquids that have phenol/chloroform must be disposed of in the "Liquid Phenol/Chloroform Waste" container found in the fume hood. None of this type of waste should be disposed of down the drain or in the regular trash.

RNA Handling: Due to the prevalence of RNases, gloves should be worn at all times when handling your samples. Samples should also be stored on ice at all times (to reduce the activity of any contaminating RNases remaining in your sample), unless otherwise noted.

Razor Blades Handling and Disposal -

• Handling - Obviously, these are extremely sharp. Use them with extreme caution. Pay careful attention to what you are cutting. Only cut tissue that is on a flat, stable surface. Do NOT attempt to cut anything with a razor blade while holding the object in your hand!
• Disposal - Razor blades MUST be disposed of in the available "Sharps" container! The "Sharps" container is bright red and easily visible. If you cannot find it, ask the TA. Under no circumstances are razor blades to be disposed of in the regular trash!

Supplies and Reagents

• micropipettes (1-1000 μL)
• sterile filter pipette tips (1-1000 μL)
• 1.5 mL microcentrifuge tubes
• microcentrifuge tube rack
• lab coats
• safety glasses
• gloves
• lab pen
• timers
• ice buckets
• phenol/chloroform waste containers (liquid/solid)
• vortex
• hot water bath
• Nanodrop spectrophotometer
• chloroform
• RNase free water
• chloroform
• isopropanol
• 75% ethanol
• 0.1% DEPC treated water

Procedure Background

This section provides an explanation of the methods being used and provieds some essential background information.

• You will isolate RNA from whole tissue using TriReagent. TriReagent allows for separation of RNA from other cellular components, including DNA. There are three primary components of TriReagent that allow this to happen. The first is guanidine isothiocyanate which is a potent protein denaturant, the second is phenol, and the third is pH.
• Guanidine isothiocyanate denatures proteins, such as the highly abundant histones that coat DNA. Even more importantly, RNases are denatured. This denaturing action allows for better access of phenol (an organic solvent) to cellular proteins and improves its ability to keep the proteins insoluble. The pH of TriReagent is acidic. The low pH keeps DNA out of solution while RNA remains soluble.
• After homogenizing/lysing your tissue in TriReagent, chloroform (another organic solvent) will be added to your sample to allow for separation of the phenol and insoluble cellular components (DNA, proteins) from soluble cellular components (RNA). This will result in three distinct layers: the organic phase (the bottom portion), the interphase (layer of cell debris) and the aqueous phase (the top portion). The aqueous phase (the RNA) can then be easily isolated.
• The RNA can be precipitated and washed to remove residual phenol and salt carryover. Then the RNA can be resuspended in a suitable solution and quantitated.
• RNA is quantitated using a spectrophotometer and measuring the absorbance of your RNA sample at 260nm (A260). The concentration of your sample is calculated with the following equation:[RNA] = 40ug/mL x A260 x Dilution Factor. The spectrophotometer we will be using calculates the concentration for us using this equation and the absorbance measurement.
• The purity of your sample can be assessed using the ratio of A260 to A230 and to A280. Various substances will absorb at 230nm, which will indicate carryover of phenol, ethanol or high salt in your sample. Proteins generally absorb light at 280nm. For clean RNA, A260/A280 should range between 1.8-2.0. The A260/A230 should range between 1.5-2.0 for clean RNA. For more information on interpreting these ratio see the Nanodrop user manual.

For a fun introduction to quantification using the Nanodrop please watch this video.

RNA EXTRACTION PROTOCOL

Continued from Lab 1

1. Turn on heating block to 55°C.
2. Incubate your homogenized tissue sample (from Lab 1) tube at room temperature (RT) for 5 mins.
3. In the fume hood, add 200uL of chloroform to your sample and close the tube. NOTE: Due to the high volatility of chloroform, pipetting needs to be done carefully and quickly. Have your tube open and close to the container of chloroform before drawing and chloroform into your pipette tip.
4. Vortex vigorously for 30s. You are vortexing correctly if the solution becomes a milky emulsion.
5. Incubate tube at RT for 5 mins.
6. Spin tube in refrigerated microfuge for 15 mins. @ max speed.
7. Gently remove tube from microfuge. Be sure not to disturb the tube.
8. Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh microfuge tube. Do NOT transfer ANY of the interphase (the white, cell debris between the aqueous and organic phase).
9. Close the tube containing the organic and interphase and properly dispose of the liquid inside the tube as well as the tube itself at the end of the lab.
10. Add 500uL isopropanol to the new tube containing your RNA and close the tube.
11. Mix by inverting the tube numerous times until the solution appears uniform. Pay particular attention to the appearance of the solution along the edge of the tube. If mixed properly, it should no longer appear viscous/"lumpy".
12. Incubate at RT for 10 mins.
13. Spin in refrigerated microfuge at max speed for 8 mins. When placing your tube in the microfuge position the tube hinge pointing up, away from the center of the microfuge.
14. A small, white pellet (RNA and salts) should be present. If not, do not fret an continue with the procedure.
15. Remove supernatant.
16. Add 1mL of 75% EtOH to pellet. Close tube and vortex briefly to dislodge pellet from the side of the tube. If the pellet does not become dislodged, that is OK.
17. Spin in refrigerated microfuge at 7500g for 5mins.
18. Carefully remove supernatant. Pellet may be very loose. Make sure not to remove pellet!
19. Briefly spin tube (~15s) to pool residual EtOH.
20. Using a small pipette tip (P10 or P20 tips), remove remaining EtOH.
21. Leave tube open and allow pellet to dry at RT for no more than 5mins.
22. Resuspend pellet in 100uL of 0.1%DEPC-H2O by pipetting up and down until pellet is dissolved.
23. Incubated tube at 55C for 5mins. to help solubilize RNA.
24. Remove tube from heat, flick a few times to mix and place sample on ice. This will be your stock RNA sample.
25. Quantitate RNA yield using Nanodrop spectrophotometer.

RNA QUANTIFICATION NOTE: Always keep your RNA samples on ice!

1. Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm.
2. Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
3. Pipette 2µL of your RNA sample onto the Nanodrop pedestal and lower the arm
4. Click "Measure". Record your RNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate RNA concentration for you. See Lab 1 notes on RNA extraction for more information on the calculation and how to evaluate RNA purity using A260/280 and A260/A230 ratios.
5. Raise the arm and wipe off you sample with a KimWipe
6. Clearly label your stock RNA sample with the word "RNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
7. Give your samples to the TA for storage at -80C.