MEGAscript RNAi Kit

Gene-specific template(s) 

A transcription template(s) is needed with T7 RNA polymerase promoters positioned to transcribe sense and antisense RNA corresponding to the target RNA. 

For dsRNA purification 

• 100% ethanol: ACS grade or better
• Equipment to draw solutions through the Filter Cartridges: Use either a microcentrifuge capable of at least 8,000 X g, or a vacuum manifold with sterile 5 mL syringe barrels mounted to support the Filter Cartridges.

A.   Choosing the dsRNA Sequence

RNAi experiments are typically done with dsRNA 400 bp and larger; 200 bp is the minimum size of dsRNA recommended for RNAi. Typically templates for transcription of dsRNA for use in RNAi correspond to most or all of the target message sequence.

B.   Strategies for Transcription of dsRNA

RNAi experiments requiredouble-stranded RNA (dsRNA). Since the T7 RNA polymerase used in this kit synthesizes single-stranded RNA (ssRNA), use one of the following strategies to produce dsRNA:

• Prepare one DNA template with opposing T7 promoters at the 5' ends of each strand, and use it in a single transcription reaction to synthesize dsRNA without a separate annealing step.
• Use two DNA templates that are identical except that a single T7 promoter sits at opposite ends of the region to be transcribed. With this strategy, the templates can both be added to a single reaction. Although both templates should be transcribed at the same rate, if they are not, the final dsRNA yield will be dictated by the template with the lower transcription efficiency. Alternatively, the two templates can be transcribed in separate reactions to make complementary RNA molecules, which are then mixed and annealed. For the annealing step, the two transcripts can be mixed in precisely equimolar amounts. Note, however, that if the templates are transcribed in separate transcription reactions, this kit contains enough reagents to produce only 10 different dsRNAs.

Figure 1. T7 Polymerase Promoter: Minimal Sequence Requirement

                                                   +1
5'-TAATACGACTCACTATAGGGAGA-3'
    |________________________|

The +1 base (in bold) is the first base incorporated into RNA. The underline shows the minimum promoter sequence needed for efficient transcription.


C.   PCR Templates

Amplification strategies to add T7 promoter sequences to DNA

T7 promoter sequences can be added to DNA using PCR to generate templates that can be directly added to the MEGAscript RNAi Kit transcription reaction. Begin by synthesizing PCR primers with the T7 promoter sequence appended to the 5' end of the primer. The T7 promoter-containing PCR primers (sense and antisense) can either be used in separate PCRs, or in a single PCR to generate transcription template for both strands of the dsRNA. The two strategies for adding T7 promoter to DNA are shown below:

Figure 1

• Calculate the annealing temperatures of the entire PCR primer (with the T7 promoter site) and the gene specific portion of the PCR primer separately.
• Since the first cycles of PCR use only the 3' half of the PCR primer(s), the gene-specific part, the annealing temperature for the first 5 PCR cycles should be ~5°C higher than the calculated T_m for the gene-specific region of the primer. We have found that using the calculated annealing temperature for the initial cycles often results in synthesis of spurious PCR products.
• Once some PCR product is made, subsequent primer annealing events (cycle 6 and thereafter) use the entire primer site; therefore use the calculated T_m for the entire PCR primer plus ~5°C for subsequent cycles.
• We recommend using primers at 100 nM in the PCR mix. Higher concentrations may result in synthesis of primer dimers. 

Check PCR products on a gel before using them in this procedure 

PCR products should be examined on an agarose gel prior to in vitro transcription to estimate concentration and to verify that the products are unique and of the expected size.


D.   Plasmid Templates

Cloning strategy 

When using plasmid templates in the MEGAscript RNAi Kit, it is best to make two separate clones with the same target region in both orientations.

Figure 2. Cloning in plasmids





Note:  Although some dsRNA templates have been cloned in plasmids containing opposing T7 promoters (on either side of the linker region Kamath 2001, Timmons and Fire 1998, Morris 2001), these constructs will result in synthesis of dsRNA with significant stretches of RNA corresponding to the polylinker region of the plasmid.

PCR products can be cloned into plasmid vectors using any of the following strategies:

• Amplify the target by PCR and ligate the product into a PCR vector with a T7 promoter. Identify plasmids with the insert in both orientations with regard to the T7 promoter.
• Or, include the T7 promoter sequence at the 5' end of one or both of the PCR primers, then perform PCR to incorporate them into the fragment. Finally, ligate the PCR product into a PCR cloning vector (one that does not include a T7 promoter).
• Or, include both a T7 promoter and a restriction site at the 5' end of one or both PCR primers to incorporate them into the fragment during PCR. Ligate the PCR product into a cloning vector using the added restriction sites.

Plasmid linearization 

Plasmid templates must be linearized downstream of the insert to create a transcription termination site—the RNA polymerase will literally fall off the end of the DNA molecule. Linearize each template, then examine the DNA on a gel to confirm that cleavage is complete. Since initiation of transcription is the rate limiting step of in vitro transcription reactions, even a small amount of circular plasmid in a template prep will generate a large proportion of transcript, wasting much of the synthetic capacity of the reaction.

Note:  We routinely use all types of restriction enzymes. However, there has been one report of low level transcription from the inappropriate template strand in plasmids cut with restriction enzymes leaving 3' overhanging ends (produced by Kpn I, Pst I, etc.; Schenborn and Mierindorf, 1985).

Figure 3  Linearized plasmids

After linearization 

Terminate the restriction digest by adding each of the following:

• 1/20 volume 0.5 M EDTA
• 1/10 volume of either 3 M NaOAc or 5 M NH4OAc
• 2 volumes of ethanol

Mix well and chill at –20°C for at least 15 min. Then pellet the DNA for 15 min in a microcentrifuge at top speed. Remove the supernatant, respin the tube for a few seconds, and remove the residual fluid with a very fine-tipped pipet. Resuspend in TE (10 mM Tris-HCl pH 8, 1 mM EDTA) at a concentration of 0.5–1 μg/μL.

Plasmid DNA purity 

DNA should be relatively free of contaminating proteins and RNA. The greatest yields of dsRNA will be obtained with very clean template preparations. Most commercially available plasmid preparation systems yield DNA that works well in the MEGAscript RNAi Kit. Alternatively, a DNA miniprep procedure that generally yields high quality template is presented in section V.D.

Note that DNA from some miniprep procedures may be contaminated with residual RNase A. Also, restriction enzymes occasionally introduce RNase or other inhibitors of transcription. When transcription from a template is suboptimal, it is often helpful to treat the template DNA with proteinase K before performing the transcription reaction.

A.   Before Using the Kit for the First Time

Prepare the Wash Solution 

1.   Add 12 mL ACS grade 100% ethanol to the bottle labeled 2X Wash Solution.

2.   Mix well, and store at room temperature.

We suggest crossing out the 2X from the bottle label after adding the ethanol. In these instructions this reagent will be called Wash Solution once the ethanol is added.

B.   Transcription Reaction Assembly

1.   Thaw the frozen reagents at room temp then place them in ice

Remove the T7 Enzyme Mix from the freezer and place it directly in ice; it is stored in glycerol and will not freeze at –20°C. Vortex the 10X T7 Reaction Buffer and the 4 ribonucleotide solutions (ATP, CTP, GTP, and UTP) until they are completely in solution. Once they are thawed, store the ribonucleotides (ATP, CTP, GTP, and UTP) on ice, but keep the 10X Reaction Buffer at room temperature. Microcentrifuge all reagents briefly before opening to prevent loss and/or contamination of any material on the rim of the tube.

2.   Assemble transcription reaction at room temperature

Assemble the transcription reaction at room temperature in the order shown below. The following amounts are for a single 20 μL transcription reaction. Reactions may be scaled up or down if desired.

Important:  The following reaction setup is recommended when the RNA produced will be ≥400 nt in length. For transcripts shorter than this, see section V.C. Optimizing Yield of Short Transcripts for modified reaction setup suggestions.

3.   Mix thoroughly

Gently flick the tube or pipette the mixture up and down, then briefly microcentrifuge to collect the reaction mixture at the bottom of the tube.

4.   Incubate at 37°C for 2–4 hr

The first time a new template is transcribed, the recommended incubation time is 2–4 hr. The optimal incubation time for a given template varies depending on its size and transcriptional efficiency. For transcripts <400 nt, a longer incubation time (up to ~16 hr) may be advantageous, since more transcription initiation events are required to synthesize a given mass amount of short RNA, compared to transcription of longer templates. Instead of doing overnight incubations, we suggest freezing the reaction overnight and then thawing and resuming the reaction the next day. Reactions are stable for several days when frozen at –20°C; however, storage will result in gradual loss of enzyme activity. To determine the optimum incubation time for maximum yield with a given template, a time-course experiment can be done. To do this, set up a transcription reaction, and remove aliquots of the reaction at various intervals (for example after 2 hr, 4 hr, 6 hr, and overnight incubation). Assess results by looking for a decrease in absorbance at 260 nm as the free nucleotides are incorporated into RNA, or by running 0.5 μL samples of the RNA on a gel stained with ethidium bromide

C.   Annealing RNA to Maximize Duplex Yield

Include this annealing step for the following types of reactions:

• All >800 nt dsRNA synthesis reactions
• ≤800 nt dsRNA synthesis reactions when the two strands were synthesized from separate transcription templates (in the same or in separate transcription reactions).

1.   Mix the transcription reactions containing complementary RNA

• If sense and antisense RNA were synthesized in separate transcription reactions, add the entire contents of one of the reactions to the other. If desired, reserve a 0.5 μL aliquot of each template before mixing for gel analysis.
• If sense and antisense RNA was synthesized in a single transcription reaction, both strands of RNA will already be in a single tube; proceed to step 2

• 1/400th of a 20 μL dsRNA solution is 5 μL of a 1:100 dilution.
• Dilute the gel samples in TE (10 mM Tris, 1 mM EDTA) or in gel loading buffer.

• a.    For each dsRNA sample, place a Filter Cartridge in a Collection Tube. Use the Collection Tubes supplied with the kit.
  
• b.    Pipet the entire 500 μL dsRNA mixture onto the filter in the Filter Cartridge. Centrifuge at maximum speed for 2 min.
  
• c.    Discard the flow-through and replace the Filter Cartridge in the Collection Tube.

• a. For each dsRNA sample, place a 5 mL syringe barrel on the vacuum manifold, load it with a Filter Cartridge, and turn on the vacuum.
  
• b. Pipet the entire 500 μL dsRNA mixture onto the filter in the Filter Cartridge. The vacuum will draw the lysate through the filter.

• a. Pipet 500 μL of Wash Solution onto the filter in the Filter Cartridge. Draw the wash solution through the filter as in the previous step.
  
• b. Repeat with a second 500 μL of Wash Solution.
  
• c. After discarding the Wash Solution, continue centrifugation, or leave on the vacuum manifold for ~10–30 sec to remove the last traces of liquid.

• a. The Elution Solution provided with the kit is 10 mM Tris-HCl pH 7, 1 mM EDTA. It is compatible with dsRNA injection, or 2X Injection Buffer can be added to the purified dsRNA for a final concentration of 1X Injection Buffer. Alternatively, the dsRNA can be eluted into any sterile low salt solution (≤30 mM), e.g. 5 mM KCl, 0.1 mM sodium phosphate buffer as used by Rubin and Spradling (1982).Transfer the Filter Cartridge to a fresh Collection Tube.
  
• b. Apply 50–100 μL (hot) Elution Solution to the filter in the Filter Cartridge.
  
  

  Apply preheated (≥95°C) Elution Solution to the filter, or
  
  Apply room temperature Elution Solution, close the tube lid over the Filter Cartridge, and incubate in a heat block set to 65°C or warmer for 2 min.

  
  
  
• c. Centrifuge for 2 min at maximum speed.
  
• d. Repeat steps b–c with a second 50–100 μL aliquot of Elution Solution collecting the RNA into the same Collection Tube. Most of the RNA will be eluted in the first elution. The second elution is included to recover any remaining RNA.

• 1/400th of 100 μL elution volume is 2.5 μL of a 1:10 dilution
  
• 1/400th of 200 μL elution volume is 5 μL of a 1:10 dilution
  
• Dilute the gel samples in TE (10 mM Tris, 1 mM EDTA) or in gel loading buffer
  
  The dsRNA will migrate slightly slower than DNA markers of the same length. 

A.    Use of the Control Template

The Control Template is a linear dsDNA fragment with opposing T7 promoters. It yields a 500 bp dsRNA product.

Positive control reaction instructions

1. Use 2 μL (1 μg) of Control Template in standard MEGAscript RNAi Kit reactions
2. Incubate the transcription reaction for 2 hr.
3. Skip section III.C. Annealing RNA to Maximize Duplex Yield  as the sense and antisense RNA strands will hybridize during the transcription reaction.
4. Follow the procedure as described in sections III.D and III.E starting on to purify the dsRNA.
5. (Optional) Run 1–2 μg of the positive control reaction product on at 1% agarose gel to verify that the dsRNA is 500 bp.

Expected yield from the control reaction

The yield of RNA from the positive control reaction should be 50–80 μg of a 500 bp dsRNA.

What to do if the positive control reaction doesn’t work as expected

If the yield of RNA from the control reaction is low, something is probably wrong with the procedure, the kit, or the quantitation.

1. Double check the RNA quantitation
   
   
   • When assessing yield by UV spectrophotometry, be sure to use TE (10 mM Tris-HCl, 1 mM EDTA) to blank the spectrophotometer and dilute the RNA.
   • To confirm that the quantitation is correct, verify the yield by an independent method. For example if UV spectrophotometry was used to assess yield, try also running an aliquot of the reaction on an agarose gel and comparing its intensity to a sample of known concentration.<
   • Consider repeating the positive control reaction. If the yield is indeed low by two different measurements, there may be a technical problem with the way the kit is being used. You may want to consider repeating the positive control reaction. If you are certain that the procedure was carried out correctly, and the control reaction does not give the expected results, contact technical support

B.    Low Yield

If the transcription reaction with your template generates full-length, intact RNA, but the reaction yield is significantly lower than the amount of RNA obtained with the Control Template, it is possible that
contaminants in the DNA are inhibiting the RNA polymerase. A mixing experiment can help to differentiate between problems caused by inhibitors of transcription and problems caused by the sequence of a template. Include three reactions in the mixing experiment using the following DNA templates:

1. 2 μL Control Template

2. 1–2 μg experimental DNA template

3. a mixture of 1 and 2

Assess the results of the mixing experiment by running 0.5 μL of the transcription reaction on an agarose gel. 

Transcription of the Control Template is inhibited by your template. (See Figure 6.a)

This implies that inhibitors are present in your DNA template. Typical inhibitors include residual SDS, salts, EDTA, and RNases. Proteinase K treatment followed by phenol:chloroform extraction frequently improves template quality. Proteinase K treatment Treat template DNA with Proteinase K (100–200 μg/mL) and SDS
(0.5%) for 30 min at 50°C, followed by phenol/chloroform extraction and ethanol precipitation. Carry-over of SDS can be minimized by diluting the nucleic acid several fold before ethanol precipitation, and excess salts and EDTA can be removed by vigorously rinsing nucleic acid pellets with 70% ethanol before resuspension.

Adding your template to the reaction with the Control Template does not inhibit synthesis of the control RNA.
(See Figure 6.b.) This indicates that the problem may be inherent to your template.

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