Lab 1: DNA isolation; initiate RNA isolation

Lab Objectives
By the end of lab you should have completed the following:
  • Select tissue for DNA, RNA and protein extraction
  • Start RNA extractions (add Tri-Reagent to samples, homogenize, and then re-freeze for next week)
  • Isolate and quantify DNA from tissue.

RNA Extraction Part 1


Supplies and Reagents
  • micropipettes (1-1000uL)
  • sterile filter pipette tips (1-1000uL)
  • sterile (RNase free) 1.5mL microcentrifuge tubes
  • sterile disposable pestles
  • vortex
  • ice buckets
  • gloves
  • lab pens
  • safety glasses
  • TriReagent

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
  • In addition to the A260, absorbance at 230nm and 280nm should be taken. The ratio of A260 to each of these absorbances can be used to assess the purity of your RNA. 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.

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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!


RNA ISOLATION PROTOCOL
  1. Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker. Keep the sample stored on ice until you are ready for homogenization.
  2. Add 500uL of TriReagent to the 1.5mL snap cap tube containing your tissue. Store on ice.
  3. Carefully homogenize the tissue using a disposable pestle. If the tissue is difficult to homogenize, carefully close the tube tightly and briefly vortex the sample.
  4. After the sample is completely homogenized, add an additional 500uL of TriReagent to the tube and close the tube tightly.
  5. Vortex vigorously for 15s.
  6. Stop here for Lab 1 and give your labeled homogenized tissue sample to the TA for storage at -80ºC. You will be finishing your RNA extraction in lab next week.

DNA Isolation (DNazol)

Supplies and Reagents
  • micropipettes (1-1000 µL)
  • sterile filter pipette tips (1-1000 µL)
  • 1.5 mL microfuge tubes
  • microcentrifuge tube rack
  • microcentrifuge (room temperature)
  • razor blades
  • vortexes
  • DNazol
  • 100% ethanol
  • 75% ethanol
  • 0.1% DEPC water
  • kim wipes
  • Nanodrop

Procedure Background
  • DNazol uses a guanadine-detergent lysing solution to hydrolyze RNA and selectively precipitate DNA from the cell. During the extraction, the cells in the sample are lysed and DNA is precipitated using ethanol. DNA is then solubilized using water or NaOH. The genomic DNA recovery should be between 70 and 100% using this protocol.

  • DNazol is an irritant, so please be careful and wear protective gear when using it.
  • Centrifuging in step 3 removes insoluble tissue fragments, partially hydrolyzed RNA, and excess polysaccharides. This step is necessary to get higher quality DNA.
  • If you have trouble removing your DNA precipitate in step 8, you can centrifuge it at 5,000 x g for 5 minutes (room temperature) so that the DNA will form a pellet.
  • We will "spec" our DNA after extraction. Purified DNA should have a A260/A280 ratio of 1.7-1.9, indicating good quality DNA.

DNazol Extraction Protocol (Adapted from MRC manual)
    1. Using a sterile pestle, homogenize your tissue sample in 0.5 mL of DNazol in a 1.5 mL sterile microfuge tube. After the tissue is homogenized, add 0.5 mL more of DNazol and mix well.
    2. Let your sample incubate for 5 minutes at room temperature.
    3. Spin your sample at 10,000 x g (room temp) for 10 minutes.
    4. Transfer your supernatant to a new, labeled tube.
    5. Add 0.5 mL of 100 % ethanol to your sample.
    6. Mix your sample by inverting your tube 5-8 times.
    7. Store your sample at room temperature for 1 minute.
    8. Your DNA should form a cloudy precipitate. Remove the DNA and put in a new tube using your pipette.
    9. Let your sample sit at room temp for 1 minute and remove the rest of the lysate (liquid that is not DNA).
    10. Wash your DNA with 1 mL of 75% ethanol: Pipette the ethanol into your DNA tube, invert 6 times, and let sit for 1 minute. Remove the ethanol from the tube and repeat.
    11. If there is ethanol left at the bottom of your tube after the second wash, remove with a small pipette.
    12. Add 300 µL of 0.1% DEPC water to your DNA and pipette up and down multiple times to dissolve.
    13. Bring your DNA sample up to the Nanodrop to quantify.

DNA Quantification

  1. Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm.
  2. Select "dsDNA" from the pulldown menu
  3. Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
  4. Pipette 2µL of your DNA sample onto the Nanodrop pedestal and lower the arm
  5. Click "Measure". Record your DNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate DNA concentration for you.
  6. Raise the arm and wipe off you sample with a KimWipe
  7. Clearly label your stock DNA sample with the word "DNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
  8. Store sample at -20ºC.