BISC 219/F10: RNAi Lab 10

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 controla and RNAi worms for your 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 treatd 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 M9 buffer and putting them in a 1.5 ml tube. Repeat for your 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 M9 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 M9 as possible 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 quickly in your hand upon removal from the freezer.

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