BISC 219/F10: RNAi Lab 10

Series 3 Reverse Genetics: Scoring RNAi Worms and RNA Isolation

3 days before next lab:

1. Come into lab and find your stack of plates.
2. On 2 of the experimental plates add 2 L4 wild type (N2) hermaphrodites
3. On 2 of the experimental plates add 2 L4 rrf-3 hermaphrodites
4. On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites
5. On 1 of the control plates add 2 L4 rrf-3 hermaphrodites
6. Wrap all of your plates in an elastic and stick in your lab day box in the worm incubator set at 23°C
   

Phenotypic Analysis

Today in lab you will examine your RNAi worms and their progeny.

1. Remove your plates from the incubator.
2. Examine your wild type (N2) and rrf-3 RNAi worms. Do you see any difference between the two strains of worms?
3. Compare your RNAi worms to the control worms - are they the same phenotype? Different?
4. Compare your RNAi worms to worms that have a known mutation in that gene (if we have them) - how do the compare?
5. Score and count approximately 50 worms per RNAi plate. What is the proportion of affected to unaffected worms?

Be sure to record all of your results in your lab notebook.
Take pictures of your control and RNAi worms to use in the results section of your next paper.

RNA Isolation

The theory of RNAi states that the RNA specfic to the gene of interest will be degraded inside the cells of the worm because double stranded RNA is not stable inside the cell. The degradation of RNA will prevent the production of the protein product of that gene and thus possibly exhibit a visible phenotype.

If your RNAi worked exceptionally well and most of your worms on your treated plate show a visible phenotype and your control worms do not we can collect all of our worms on the plate by washing them off with 1 ml of 1X PBS + Tween 20 buffer and putting them in a 1.5 ml tube. Repeat for your untreated control worms in a separate tube.

If less than 80% of your worms show a visible phenotype then pick 100 worms into 100 μL of 1X PBS + Tween 20 buffer in a 1.5 ml tube. Repeat for your control worms in a separate tube.

In both cases let the worms settle to the bottom of the tube and remove as much of the PBS as possible with a pipette without sucking up worms before moving on to the next step.

In order to break through the tough cuticle we must freeze/thaw the worms. Place the worms in minimal amounts of M9 buffer in the -80°C freezer for 15 minutes. Thaw them quickly by wrapping your warm hand around the tube just after you remove them from the freezer.

The next steps require the use of Phenol and Chloroform - please wear gloves and work in the hood! Phenol will burn your skin on contact.

1. Add 500 μL Trizol (Invitrogen #15596-026) to both control and treated worms.
2. Incubate at room temperature for 15 minutes (dissociates the nucleoprotein complexes)
3. Add 100 ul of Chloroform and make sure the cap is snapped on well.
4. Shake vigorously for 15 seconds.
5. Incubate 2 minutes at room temperature
6. Spin at 12,000g for 15 minutes at 4°C
7. After spinning the top aqueous phase contains the RNA, the interphase (cloudy) contains the DNA and the lower red phase contains the proteins. Transfer the clear top aqueous phase to a clean 1.5 ml microfuge tube. BE VERY CAREFUL NOT TO GET THE DNA INTERPHASE - IT WILL MESS UP YOUR RESULTS!
8. Add 250 μL Isopropanol to each reaction, mix by inversion a few times and incubate at room temperature for 10 minutes. This will begin the RNA precipitation.
9. Spin at 12,000g for 15 minutes at 4°C
10. Remove the supernatant and look for a pellet on the side of your tube - you may or may not see one.
11. Wash the pellet with 75% Ethanol. Slowly add to the tube and then draw back off and discard. If you pellet is floating spin the tube before removing the wash.
12. Turn the tube upside down on a kimwipe and let air dry for 5 minutes.
13. Add DEPC treated water - see your instructor for volume (20-50 μL likely).

DNase Treatment

To be sure that the only nucleic acid you will amplify in your RT PCR reaction is RNA and not contaminating DNA we will treat our samples with an enzyme DNase. We will use TURBO DNase from Applied Biosystems. It is a genetically modified and more efficient form of DNaseI.

1. Measure the nucleic acid concentration using the NanoDrop2000. See your instructor.
2. If your concentration is >200 ng/ul dilute your sample so that it is 200 ng/ul.
3. Add 10X Turbo DNase buffer to make a final concentration of 1X in your sample. Show your calculations to your instructor before this step.
4. Add 1 ul of TURBO DNase (2 units) to each sample.
5. Incubate at 37°C for 30 minutes.
6. Add EDTA to your sample to a final concentration of 15 mM
7. Incubate at 75°C for 10 minutes to heat kill the DNase.
8. Determine your nucleic acid concentration again using the NanoDrop. Record ALL information in your notebook for next week.

RNAi General Information
Media Recipes
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 11: RT PCR reactions