---
# System prepended metadata

title: RNA Synthesis

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# RNA Synthesis

[EcoEvoDevo Lab](https://hackmd.io/@aphanotus/AngeliniLab)

![](https://i.imgur.com/J02r7aM.jpg)
> By brewbooks from near Seattle, USA - Grand Prismatic Spring, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=51511238

## General considerations

RNA synthesis allows RNAi and CRISPR. The preparation of dsRNA or sgRNA from a template DNA sequence has 4 steps. First, a region within the template DNA is amplified by PCR using primers that add the T7 RNA polymerase promoter sequence to one or both ends. Second, that PCR product is used as template in an in vitro transcription reaction. After annealing, the new RNA is purified. Finally, the RNA concentration is quantified and prepared for microinjection. 

### Working with RNA

Single-strand RNA is prone to degradation by RNase, which can be ubiquitous in the environment. As you prepare RNA, take precautions that will preserve your efforts. 

- Keep reagents cold. Let tubes thaw on ice, and keep your reactions on ice until they are ready for incubation at higher temperatures. 
- Always use filtered pipette tips to prevent introduction of RNase
- Be sure that water used in your reactions is reagent-grade and nuclease-free.
- Wear clean gloves. Change them if you do something that may expose your hands to RNase, like if you touch a computer, phone or notebook.
- Keep tubes containing your reagents and reactions open as breifly as possible. Don't breathe heavily over them!

## PCR to create a template for RNAi

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. 

The template can be either a purified plasmid containing a fragment of the gene's sequence (miniprep) or a linear synthetic DNA (gBlock). Because these reagents contain the target DNA sequence *only*, just a trace amount is needed to template the PCR reaction. If you prefer, you attempt to pipette 0.1 μl of miniprep of gBlock DNA into the reaction.

- Combine the following reagents in a 200-μl PCR tube:

|        |                    |
| ------:|:------------------ |
| 6.0 μl | [JumpStart Taq Mix](https://www.sigmaaldrich.com/catalog/product/sigma/d9307)  |
| 0.5 μl | forward primer  |
| 0.5 μl | reverse primer |
| trace | template DNA |
| 5.0 μl | water (nuclease-free) |
| 12.0 μl | total |

- Mix the solution by pippetting up and down. (Set a pipetter to 3 μl, then gently pipette the solution up and down a few times. Don't intentionally remove any volume.)
- Close the tube and label it.
- Run a thermocycler program with the following conditions. (This should take about 45 minutes.)

|      |        |              |
| ---- |:------ |:------------ |
| 98˚C | 2 min  |              |
| 98˚C | 10 s   | \|           |
| 50˚C | 30 s   | \| 35 cycles |
| 72˚C | 30 s   | \|           |
| 72˚C | 2 min  |              |
| 12˚C | hold   |              |

On our lab's C1000 thermocycler, use the program called `T7 template`.

## PCR to create a template for an sgRNA

- Combine the following reagents in a 200-μl PCR tube:

|        |                    |
| ------:|:------------------ |
| 6.0 μl | [JumpStart Taq Mix](https://www.sigmaaldrich.com/catalog/product/sigma/d9307)  |
| 0.5 μl | gene-specifc primer  |
| 0.5 μl | universal-Cas9sg-R primer |
| 5.0 μl | water (nuclease-free) |
| 12.0 μl | total |

- Mix the reaction by pippetting up and down. (Set a pipetter to 3 μl, then gently pipette the solution up and down a few times. Don't intentionally remove any volume.)
- Close the tube and label it.
- Run a thermocycler program with the following conditions. (This should take about 45 minutes.)

|      |        |              |
| ---- |:------ |:------------ |
| 98˚C | 10 s   | \|           |
| 60˚C | 30 s   | \| 35 cycles |
| 72˚C | 15 s   | \|           |
| 72˚C | 10 min |              |
| 12˚C | hold   |              |

## Q/C

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

## In vitro transcription

Next we will use a modification of the MEGAscript T7 Transcription Kit (Life Technologies [item AM1334](https://www.thermofisher.com/order/catalog/product/AM1334)). The nucleotide triphosphates (NTPs) arrive in individual tubes. These should be combined into a new tube with equal volumes (and concentrations) of each. 

- Set up the T7 transcription reaction as follows in a 200-μl tube:

|      |                     | 
| ----:|:------------------- | 
| 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 the reaction by gently pippetting up and down. 
- Incubate the reaction at 37˚C for 1-4 hours, using the incubation mode on the thermocycler. (A range of 1-16 hours seems to work. The optimum appears to be 4 hours.)
- When the incubation is done, without cancelling the run, open the machine and remove the reaction tubes.
- Add 1 μl of [TURBO DNase](https://www.thermofisher.com/order/catalog/product/AM2238), 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 complementary RNA to adopt its proper secondary structure.

|      |                     | 
| ----:|:------------------- | 
| 95˚C | 3 min | 
| -0.1˚C/s ramp | takes about 9 min | 
| 45˚C | 1 min | 
| 12˚C | hold | 

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

## RNA purification

- Add 50 μl of 7.5M ammonium acetate and 300 μl 100% ethanol. Both these reagents should be stored at -20˚C until use.
- Vortex the sample.
- Precipitate the RNA at -20˚C for at least 20 min (or up to 2 days).
- 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 30 μl (suggested) of nuclease-free low Tris-EDTA Buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
- Use the NanoDrop spec to measure the concentration and purity of the RNA. Record this information in your notebook.  Concentrations should be at least 1 μg/μl, but may vary depending on the length of time allowed for RNA synthesis. Pure RNA should have an A~260~/A~280~ ratio greater than 2.0.
- To confirm the quality of the RNA, run out 1 μl on a 2% agarose gel. It's convenient to run the RNA beside the template PCR product. Double-strand RNA should run very close to DNA of the equivalent sequence and length. However, single-strand RNA will not run to the same distance as double-strand DNA of the same length. Bands much higher than the template DNA represent complex RNA secondary structures that are typically unsuitable for RNAi.
- Based on the measured concentration of RNA, dilute the solution as follows. (Use the old dilution equation, *c~1~v~1~ = c~2~v~2~*. Start with an initial volume, *v~1~*, that estimates the total volume of sgRNA solution available.)
  - for dsRNA: 2000 ng/μl in nuclease-free water
  - for sgRNA: 500 ng/μl in nuclease-free low Tris-EDTA buffer 
- Aliquot 5 μl into 0.5-ml tubes.
- Store at -80°C.

## Preparing buffered dsRNA solutions for RNAi

- When making the dilution of dsRNA, include a dilution of 100X injection buffer to the working concentration (1X), and a 1:20 dilution of McCormick green food coloring or 0.5% [phenol red](https://en.wikipedia.org/wiki/Phenol_red) solution

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, c~1~v~1~ = c~2~v~2~, where c~1~ and c~2~ are the initial and final dsRNA concentrations (which you know), v~1~ and v~2~ 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:

c~1~v~1~ = c~2~v~2~

(1892.5 ng/μl) v~1~ = (1000 ng/μl)(25 μl)

v~1~ = (1000 ng/μl)(25 μl) / (1892.5 ng/μl)

v~1~ = 25000 μl / 1892.5

v~1~ = 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. 

## Preparation of Cas9/sgRNA solution

### Cas9 prep

#### As described by [Martin et al. 2020](https://jeb.biologists.org/content/223/Suppl_1/jeb208793.long).

- Resuspend 65 μg NLS-Cas9 ([QB3 Berkeley](http://qb3.berkeley.edu/macrolab/cas9-nls-purified-protein/)) in 45 μl RNAse-free water. 
- Add 10 μl 0.5% [phenol red](https://en.wikipedia.org/wiki/Phenol_red) solution
- Aliquot 5 μl into 0.5-ml tubes
- Store at -80°C

#### Modified

The NLS-Cas9 arrived in 10 μl aliquots of a solution at 6.4 μg/μl. I diluted this to approximate the concentration specified above using the steps below.
- Add 34.3 μl of nuclease-free water to one Cas9 aliquot.
- Add 9.8 μl of 0.5% phenol red.
- Mix.
- Place 5-μl aliquots in 0.5 ml tubes.
- Store at -80˚C.

#### Combine Cas9 and sgRNA immediately before use

Guide RNAs and Cas9 should be combined just prior to [microinjection](https://hackmd.io/@aphanotus/microinjection).

- Thaw 5 μl of Cas9 stock and 5 μl of sgRNA on ice
- Combine and add 10 μl RNAse-free water
- Mix gently by pipetting
- Incubate at room temperature for about 10 min, then keep on ice until use

Final concentrations should be about 250 ng/μl Cas9, 125 ng/μl sgRNA, and 0.02% phenol red.

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[EcoEvoDevo Lab](https://hackmd.io/@aphanotus/AngeliniLab)

