Series 3 Reverse Genetics- RT PCR
You have seen, we think, the phenotypic result of RNAi mediated down-regulation or silencing of a C. elegans gene in N2 or rrf-3 worms that have a perfectly normal phenotype when not fed bacteria encoding worm-gene dsRNA. You have isolated and purified RNA from those RNAi treated and untreated control worms. The ONLY difference between the control and RNAi treated worms was the exposure to bli-1 dsRNA in the treated worms; therefore, our assumption is that the phenotypic differences we saw were due to down-regulation of bli-1 expression by the mechanism of interference RNA (RNAi). How can we be more certain that this down-regulation of gene expression was RNAi (post-transcriptionally) mediated? The first step in determining if the phenotypic abnormality you saw in the treated worms worms was likely to be because of a diminished number of mRNA transcripts for bli-1 gene product, is to reverse transcribe (RT) the mRNA of both the treated and untreated worms into copy DNA (cDNA). Copy DNA is not usually identical to the DNA sequence found on the chromosome because the template for this form of the gene is mRNA. These transcripts are not identical to their chromosomal DNA templates because of the many steps in processing that happen before the transcripts leave the nucleus. One of the most important parts of this processing is to remove non-coding regions (introns). There is a viral enzyme called reverse transcriptase that is able to make cDNA from mRNA. Once we have made cDNA from our mRNA, we can use that cDNA as a template to amplify our C. elegans gene of interest using bli-1 specific primers in a PCR reaction.
There are a few ways you can make cDNA from mRNA and they all involve a specific type of primer. In decreasing order of specificity, 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. 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 and attempt to isolate mRNA from our total purified RNA.
We will be following the protocol in the Invitrogen SuperScript™ First-Strand Synthesis System for RT-PCR (Cat. No: 11904-018).
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
|| 5 μL
| 10 mM dNTP mix
|| 1 μL
| Primer (0.5 ug/ul oligo(dT)12-18
|| 1 μL
| DEPC treated water
|| 3 μL
2. Add each component to 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
6. Add 9 μL of the 2X reaction mix to each RNA/primer mixture
This 2x reaction mix contains:
| Component || Amount
| 10X RT buffer - 200 mM Tris-HCl (pH 8.4), 500 mM KCl
|| 2 μL
| 25 mM MgCl2
|| 4 μL
| 0.1 M DTT
|| 2 μL
| RNaseOUT™ 40 U/ul
|| 1 μL
7. Mix gently and centrifuge
8. Incubate at 42°C for 2 minutes
9. Add 1 μL of 50 unit/ul 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 μL of 2 unit/ul RNase H and incubate at 37°C for 20 minutes - this digests any mRNA left in the reaction
Proceed directly to the PCR (polymerase chain reaction) to amplify your gene of interest from template cDNA.
Each master mix includes:
31 μL 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
| Gene name || Forward Primer || Reverse Primer
|| 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'
| Step || Temperature || Time || Repeat
|| 2 minutes
|| 1 time
|| 30 seconds
|| 30 seconds
|| 1.5 minutes
|| Steps 2-4 30 times total
|| 10 minutes
|| 1 time
|| 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. 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 = 1072 bp
What will you learn from finding out that your amplified gene is or is not of equal concentration in treated and untreated control worms? REMEMBER our main goal is to be sure that the aberrant phenotype that we observed in these N2 or rrf-3 (NORMAL) worms is due to POST-TRANSCRIPTIONAL gene expression regulation because the C. elegans gene was silenced or knocked down by RNAi and not because of a problem with 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
- Amplify gene of interest by pcr ;
- Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
- Clean up DNA (remove enzymes);
- Cloning: ligate gene into vector plasmid with amp resistance gene ;
- Transform competent bacterial cells;
- Select for transformants on media with ampicillin;
- Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
- Culture the selected colony from colony pcr to create a lot of copies of these bacteria
- Isolate the cloned plasmid DNA from that cultured colony by miniprep;
- Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
- Select for transformants on media with ampicillin
- Choose an isolated colony to culture and make lots of feeder strain bacteria;
- Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction.
- 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)to amplify the C. elegans gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.
Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.
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.