RNA extraction

EcoEvoDevoLab
Angelini Lab, Department of Biology, Colby College

Preparation of dsRNA

dsRNA synthesis

The preparation of dsRNA from a cloned gene sequence has 4 steps. First, a region within the cloned sequence is amplified by PCR using primers that add the T7 promoter sequence to both ends. Second, the T7 PCR product is used as template in a bi-directional in vitro transcription reaction. After annealing, the new dsRNA is purified. Finally, the dsRNA concentration is quantified and diluted in buffer for microinjection.

• Remember, single-strand RNA is prone to degradation by RNase. As you prepare dsRNA, take precautions that will preserve your efforts.
• Wear clean gloves. Change them if you do something that may expose your hands to RNase, like type on a computer, or touch a doorknob.
• Always use filtered pipette tips.
• Be sure that water used in your reactions is recently-opened, reagent-grade, Znuclease-free water.

PCR to produce a T7 template

The unique feature of this PCR is the primers. The 3' half of each primer consists of 20-nt specific to the known sequence of your target gene. The primers should amplify a region from 25-400 bp, with 200-bp being typical. The target region should avoid conserved domains. If possible, no 21-bp window within the amplified sequence should occur anywhere else in the species' transcriptome. This primer design will help avoid off-target RNAi effects.

The 5' half of each primer must consist of the 20-nt sequence of the T7 viral promoter, taatacgactcactataggg. This sequence will specify the start of transcription for the T7 RNA polymerase used in the next phase.

Set up a PCR reaction using Ex Taq, or whatever work-a-day Taq is available:

For Regular Taq

Volume	Reagent
1.25 μl	buffer (10X)
1 μl	dNTPs (10 mM each)
0.5 μl	forward primer (5 μM)
0.5 μl	reverse primers (5 μM)
0.1 μl	Taq polymerase
trace	template plasmid DNA
9.15 μl	water (distilled, nuclease-free)
12.5 μl	(Total)

For JumpStart Taq Mix

Volume	Reagent
6 μl	JumpStart Taq Mix
0.45 μl	forward primer
0.45 μl	reverse primer
trace	template plasmid DNA
5.1 μl	water (distilled, nuclease-free)
12.5 μl	(Total)

Because the template of the PCR reaction will be a highly concentrated plasmid (a miniprep product), only a trace amount is necessary as the template when setting up this reaction. Do it this way…

• Set the pipetter to 2-5 μl, then pipette the miniprep up and down a few times. Don't intentionally remove any volume.
• Using the same tip, pipette up and down in the reaction mix. The trace amounts of plasmid on the tip should provide adequate template.
• Be sure to mix your PCR reactions well, label, and cap tightly.

The PCR cycling conditions are relatively standard and these reactions typically work well since the template is highly concentrated and the primers match exactly in their 3' 20 nt.

• On the C1000 thermocycler, use the program called T7 template.

step	Temperature	Duration
1	98˚C	2 min
2	98˚C	10 s
3	50˚C	30 s
4	72˚C	30s
5	72˚C	2min
6	12˚C	hold

Repeat step 2-4 35 times

• Confirm the intended product size by running the products on a gel. There should be a single, strong band.

In vitro transcription

We use a modification of the Ambion MEGAscript T7 Transcription Kit (Life Technologies catalog number AM1334). The nucleotide triphosphates (NTPs) arrive in separate tubes. These should be combined into a new tube with equnal volumes of each. If you need large amounts of dsRNA, this reaction can be scaled up.

• Set up the transcription reaction as follows in a 200 μl tube:

Volume	Reagent
7 μl	nuclease-free water
8 μl	NTPs
2 μl	reaction buffer (10X)
1 μl	template PCR product
2 μl	T7 RNA polymerase enzyme mix
20 μl	(Total)

• Mix well by pipetting up and down.
• Incubate the reaction at 37˚C for 3-4 hours using the incubation mode on the C1000 thermocycler. (A range of 1-16 hours may also work.)
• When the incubation is done, without cancelling the run, open the machine and remove the reaction tubes.
• Add 1 μl of TURBO DNase, mix well, and return the tubes to incubation at 37˚C for 15 min.
• Cancel the incubation and run the program "Anneal dsRNA", which denatures the RNA and inactivates the DNase by heating it, then cools slowly to allow the complementary RNA to anneal into dsRNA.

Tempearture	Column 3
95˚C	3 min
0.1˚C/s	slow ramp; takes about 9 minutes
45˚C	1 min
12˚C	hold

• Add 79 μl of nuclease-free water.1
• Transfer the solution to a nuclease-free 0.5 ml tube.

RNA purification by precipitation in ammonium acetate and ethanol

This is an old school molecular biology methods that can be used to purify anything nucleic acid that's in a relatively high starting concentration. The volumes listed below assume a 100 μl initial volume. Store the ethanol and ammonium acetate at -20˚C before use.

• Add 50 μl of cold 7.5M ammonium acetate and 300 μl cold 100% ethanol.
• Vortex the sample.
• Incubate at -20˚C for at least 20 min.

This cold incubation may be continued for up to 2 days. Longer incubation will reduce yield.

• Centrifuge for 20 min at 12,000 rpm at 4˚C. Discard the supernatant.
• Wash the pellet with 0.5 ml cold 70% ethanol.
• Centrifuge for 5 min at 10,000 rpm at 4˚C. Discard the supernatant.
• Dry the pellet in a vacuum centrifuge ("speed-vac") on the medium heat setting. Be sure to leave the caps open to allow evaporation. This should take about 10-15 minutes. Stop before the pellet becomes an opaque white color.
• Resuspend the RNA pellet in 50 μl (suggested) of nuclease-free water.
• Heat the tubes for no more than 2 minutes on a heat block set no higher than 50˚C. After vortexing samples, the pellet should be completely dissolved.
• Use the NanoDrop spec to measure the concentration and purity of the dsRNA. Record this information in your notebook. Record the concentration on the side of the tube. Concentrations should be at least 1 μg/μl. Pure RNA should have A260/A280 and A260/A230 ratios greater than 2.0
• To confirm the double-stranded structure of the RNA, run out 1 μl on a 1% agarose gel. It's convenient to run the RNA beside the template T7 PCR product. Double-strand RNA should run very close to DNA of the equivalent sequence and length. Do not proceed with injections unless your RNA passes this test!
• A band half the size of the template DNA is un-paired single stranded RNA. Bands much higher than the template DNA represent complex RNA secondary structures that are also unsuitable for RNA interference.
• Store dsRNA at -20˚C.

Dilution and buffering of dsRNA for injection

• Based on the measured concentration of dsRNA, dilute the solution to a standard concentration, such as 2 μg/μl (or 2000 ng/μl).
• Use the old dilution equation, c1v1 = c2v2. You can start by arbitrarily choosing a final volume, such as 25 μl.
• When making the dilution of the dsRNA, include a dilution of 100X injection buffer to the working concentration (1X), and a 1:20 dilution of McCormick green food coloring.
• Store dsRNA at -20˚C. dsRNA should be used within 3 months.

An example of the math required to make a dilution of dsRNA

Suppose you successfully made a dsRNA solution that is resuspended in 50 μl water. The NanoDrop tells you the concentration is 1892.5 ng/μl. The ratios are all above 2.0 and the gel shows that the RNA runs to the same distance as the template PCR product (so it's double-stranded). What next?

• Choose a final concentration. Because the dsRNA solution's concentration is below 2000 ng/μl, so cannot dilute it to make a solution of that concentration. So, you can choose to make a solution of 1500 or 1000 ng/μl. (If you really need a more concentrated solution, you can use the speed-vac to partially reduce the volume of the dsRNA solution, then spec it again.)
• Choose a final volume. You have a little less than 50 μl. As long as the concentration of the starting solution isn't very close to your target, you should be able to choose any final volume less than what you have already. It's also smart to save some of the undiluted dsRNA solution for future use. For this example, let's make a 25 μl dilution.
• Calculate how much buffer and dye you must add. These reagents always start at the same concentrations. Injection buffer stock is 100X. So it must be diluted 1:100 to have a 1X working concentration in your final solution. If we want a 25 μl final volume, then the volume of 100X buffer that must be added is (25 μl)/100 = 0.25 μl. Similarly, the green food coloring dye is 20X working concentration. So we must add (25 μl)/20 = 1.25 μl.
• Calculate how much dsRNA you must add. Use the equation, c1v1 = c2v2, where c1 and c2 are the initial and final dsRNA concentrations (which you know), v1 and v2 are the initial and final volumes. You've decided the final volume will be 25 μl, so a little algebra will give you the volume of the initial solution to dilute. For our example:

c1 v1 = c2 v2
(1892.5 ng/μl) v1 = (1000 ng/μl)(25 μl)
v1 = (1000 ng/μl)(25 μl) / (1892.5 ng/μl)
v1 = 25000 μl / 1892.5
v1 = 13.21 μl

• Calculate how much water you must add. Nuclease-free water will simply make up the remaining volume to get to 25 μl. In this example, the other reagents will take up 0.25 μl (buffer) + 1.25 μl (dye) + 13.21 μl (dsRNA) = 14.71 μl. That leaves 10.29 μl for water.
• Record all these numbers in your lab notebook. If you're preparing many dsRNA solutions you can list these details in a table. The numbers from our example might look like the second line below.