BISC 219/F10: RNAi Lab 11

Series 3 Reverse Genetics- RT PCR

You are now ready to do the experiment we have been working on over the past few weeks. You have isolated and purified RNA from your RNAi treated and control worms. The first step in determining if the worms that exhibited a phenotype did so because of a diminished amount of RNA or something else, is to reverse transcribe (RT) our mRNA into cDNA (copy DNA - DNA that is lacking introns as it was made from processed mRNA). The second step uses the cDNA as a template for a PCR reaction.

There are a few ways you can make cDNA from mRNA and they all involve a specific type of primer. You can use 1) a gene specific primer, 2) an oligo(dT)_12-18 primer which binds to the polyA tail of mRNA or 3) random hexamer primers which bind to ALL RNA. What your desired outcome is will determine which primers to use. If you want to do multiple different reactions from the same RT reaction then you should consider using the oligo(dT)_12-18 primer or the random hexamer primers. The oligo(dT)_12-18 primer binds to the polyA tail found on the end of eukaryotic mRNA's. The mRNA comprises only 1-2% of the RNA in the cell. Random hexamers bind to all RNA in the cell and thus are the most non-specific primers to use.

For our reactions we will use the oligo(dT)_12-18 primer for the RT reaction.

We will be using 0.2 ml tubes (small PCR tubes) for our reactions.

1. Mix and briefly centrifuge each component before use

Component	Amount
RNA	ul
10 mM dNTP mix	1 ul
Primer (0.5 ug/ul oligo(dT)12-18	1 ul
DEPC treated water	to a total of 10 ul

2. Combine each component into the tube
3. Incubate the RNA/primer mixture at 65°C for 5 minutes
4. Place sample on ice for at least 1 minute
5. Obtain the premixed 2X reaction mix from your instructor

Component	Amount
10X RT buffer	2 ul
25 mM MgCl2	4 ul
0.1 M DTT	2 ul
RNaseOUT™ 40 U/ul	1 ul

6. Add 9 μL of the 2X reaction mix to each RNA/primer mixture
7. Mix gently and centrifuge
8. Incubate at 42°C for 2 minutes
9. Add 1 ul of SuperScript^™ II RT to each tube
10. Incubate at 42°C for 50 minutes
11. Terminate the reaction at 70°C for 15 minutes.
12. Chill on ice
13. Collect by centrifugation
14. Add 1 ul of RNase H and incubate at 37°C for 20 minutes

Proceed directly to the PCR (polymerase chain reaction) to amplify your template cDNA.
Each master mix includes:

     31 ul of dH₂O
      5 μL of 10x PCR buffer
      1 μL of 10 mM dNTPs
      1 μL of forward primer (20 μM stock)
      1 μL of reverse primer (20 μM stock)
      1 μL of 5 units/μL Taq Polymerase
Total PCR reaction volume = 50 μL (10 μL RT reaction + 40 μL PCR Master Mix

Primer sequences:

Gene name	Forward Primer	Reverse Primer
bli-1	5'ATG CAT AGA TCT CAC ACC GAC AAA CTC CAC ACG AGT TGT A 3'	5'ATG CAT ACT AGT TTA TTA TGG CAA GTG GGG GAA GGG GTG A 3'
lon-2	5'ATG CAT AGA TCT TTT GCA CAC ACC AAG ACC ATG CAC TCC C 3'	5'ATG CAT AAG CTT GAT TTA GGC GAT TTC CTT GAC TGG AAT C 3'
vab-10	5'ATG CAT ACT AGT ATG ATG GAG AAT CGG AAG AAA CCA TCT 3'	5'AGA TCT GAT TTT GTT CAT GGC AAT CGT GTT GGT C 3'
rol-5	5'ATG CAT AGA TCT GAT TAT CTG CTC CAC CAG GTC AAC CAC C 3'	5'ATG CAT AAG CTT TTA GAT ATT TCT GTA TCC ACG G 3'

PCR Conditions:

Step	Temperature	Time	Repeat
1	94°C	2 minutes	1 time
2	94°C	30 seconds
3	54°C	30 seconds
4	72°C	1.5 minutes	Steps 2-4 30 times total
5	72°C	10 minutes	1 time
6	4°C	forever	end program

Agarose gel electrophoresis of PCR product:
(NOTE: Gels might be run by the instructors if you run out of time after performing the RT reaction and the pcr amplification.)
Prepare a sample for electrophoresis by loading 10 μL of your PCR product with 1-2 μL of loading dye on a 1.0% agarose gel with SybrSafe™ stain. Run the gel at 100V for 45 minutes to an hour. The gel will be photographed using UV light and the photo posted to the lab conference.

The expected DNA fragment size amplified using the primers described above are:
bli-1 =
rol-5 =

What will you learn from finding out that your gene is or is not the expected size? REMEMBER our main goal is to be sure that the aberrant phenotype that we observed in these rrf-3 (NORMAL) worms is because bli-1 or rol-5 gene was silenced or knocked down and not due to a mutation in the gene.

Outline of Experimental Design for REVERSE Genetics Project

Where are you now in this process?(What have you done so far; What's next?)
Make the feeder strain of bacteria

1. Amplify gene of interest by pcr ;
   
2. Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
   
3. Clean up DNA (remove enzymes);
   
4. Cloning: ligate gene into vector plasmid with amp resistance gene ;
   
5. Transform competent bacterial cells of a strain genetically modified to be tetracycline resistant;
6. Select for transformants on media with tetracycline and ampicillin;
   
7. Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
8. Culture the selected colony from colony pcr to create a lot of copies of these bacteria
9. Isolate the cloned plasmid DNA from that cultured colony by miniprep;
   
10. Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
    
11. Select for transformants on media with tetracycline and ampicillin
12. Choose an isolated colony to culture and make lots of feeder strain bacteria;
    
13. Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction.
    
14. Seed NM lite worm growth media plates with feeder strain produced as described
    

Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).

Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.

Isolate RNA from RNAi worms and control worms of same strains.

Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.

Visualize cDNA in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of coding regions of gene of interest.

Assignment

Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.

Links to Labs& Project Info

Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Series3:
Schedule of Reverse Genetics Project
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 10: Scoring your worms and RNA purification
Lab 11: RT PCR reactions