# cDNA Synthesis

[EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)
*Angelini Lab, Department of Biology, Colby College*
Updated November 29, 2016 by Dave Angelini

## RNA extraction & cDNA synthesis

### General Considerations
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#### Basics of Biochemistry
During most of your molecular work, you won't be able to see the DNA, RNA or proteins you are manipulating. Therefore, it is helpful to keep in mind basic principles of biochemistry that should provide you with "common sense" for how to handle most of these procedures.
* For example, all molecules are prone to degradation through reactions with water or other reagents. This is slowed by cold, so it makes sense to keep reagents cool whenever possible.
* Most biological macromolecules are also excellent food sources for bacteria. While truly sterile technique isn't usually needed, try to keep tubes, tips, other plasticware and reagents covered as much as possible. For this reason our lab uses filtered, sterile pipette tips for most applications.
* DNA is very durable. It can usually be heated and vortexed without degradation. For this reason DNA can also cause contamination of other samples if it is handled sloppily.
* RNA is unstable. It's 2'-hydroxyl group accelerates its chemical degradation, and bacteria (and human cells) produce RNase that digest RNA. Be extra careful to avoid contamination with RNA. RNA is however also tolerant of heat.
* Proteins are also unstable. Never freeze (at -20˚C) a protein unless it is in a solution of at least 10% glycerol. Never vortex a solution with a protein that needs to be functional. It will cause a messy froth, but more importantly the protein can be denatured at the air-water interface.
* Many small molecules, like (d)NTPs are also unstable.
* Salts, buffers, and many common organic solvents, like ethanol, DMSO, and glycerol are generally very stable. However, in certain combinations they may interact.

#### Be Organized
There's an old joke: Why do they call it "research"? -- Because you have to repeat it.

You will likely undertake many similar reactions in your time in the lab, so being able to identify individual reaction products in a freezer box months later will be important. **Keep careful records in your lab notebook**. (Refer to the SOP on lab notebooks.) Always give a brief explanation of why you're doing each procedure and what you hope to achieve from it. Critically, your notebook must be dated and list the ID numbers of the samples you generate.

The standard method our lab uses for identify samples created through molecular methods, like PCR, is to use your initials, a unique number, and the date. So, the first reaction I generate today, May 30, would be "**DA#1/5-30**". It is also important to provide some explanation of what the sample is. PCR products can go in a box labeled "PCR products" in the refrigerator (4˚C). But other, less common sample types should be labeled appropriately. For example "Of total RNA", "Jh L5 male cDNA", etc. If a concentration is measured, that value should be recorded in your notebook and on the side of the sample tube.
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### Sample storage and homogenization
You will need to isolate RNA from animal tissue in order to make cDNA for the isolation of new gene sequences, for real time PCR measurement of gene expression, for transcriptome sequencing, and other applications. After homogenization there are many ways to isolate nucleic acids.
#### Materials
* Pestles for 1.5 ml tubes – VWR catalog number KT749520-0000
* RNAlater (optional)
* Lysis buffer appropriate to your method of RNA isolation
#### Procedure
* **Weigh the tissue** that you will use for RNA extraction. If you’re starting from live bugs, try not to aggravate them—that may alter their gene expression. If you’re starting from dissected tissue, keep it as cold as possible (on ice, or it can be flash frozen with liquid N~2~) or in RNAlater. Knowing the mass of tissue you start with helps calculate yield and the volume of lysis buffer needed.
* **Transfer the tissue** to a conical 1.5 ml microfuge tube. The light blue tubes are manufactured to fit with the dark blue pestles. Use only clean, RNase-free tubes.
* **Freeze the specimens** by placing live insects at -80˚C for at least 5 minutes.
* For high-sensitivity applications, like qPCR, if the specimen will be stored at -80˚C for any longer than about 12 hours, then it should be immersed in RNAlater. For RNAlater to work, the exoskeleton must be breached. So, dissect the target tissue or bisect the specimen.
* **Prepare the lysis/homogenization buffer.**
* For the PureLink Mini Kit: 198 μl lysis buffer, 2 μl β-mercaptoethanol per 10 mg.
* For Maxwell simplyRNA Kit: 40 μl homogenization buffer per 10 mg, up to a maximum of 200 μl per sample.
* Add the buffer to the frozen tissue. **Pulverize the tissue** with a pestle in the bottom of your tube. You may still see bug bits like legs, but do your best to reduce the sample to a fine gray powder. Work as fast as possible, while being safe.
* Save and wash the pestles in bleach. If the lab is ever poor, we can re-use these.
* For Maxwell isolations, samples may be stored frozen at -80°C after homogenization. Thaw homogenates on ice or at 4˚C to avoid RNA degradation.
* **For bugs** and other small insect tissues, **centrifuge** for 2 min at 12,000 ×g. Discard the pellet. (The supernatant contains the RNA.)
* **For bumblebees** and other insects with tough exoskeletons or large amounts of pile (fur), after thorough pulverization, **centrifuge** for 5 min at 20,000 ×g. Move the supernatant to a new tube. (Discard the pellet.) **Centrifuge** for 5-10 min at 20,000 ×g. Carefully remove the supernatant without disturbing the pelleted pile or any gooey material at the surface.
* Homogenization of these insects will necessarily leave behind a large volume of material. Therefore, if you're using the Maxwell isolation method (recommended) it is possible to start with more than 200 μl of homogenization buffer. However, the cartridges will not accommodate more than 200 μl of supernatant.
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### RNA isolation using column centrifugation
#### Materials
* Ambion PureLink RNA Mini Kit – Life Technologies catalog number 12183018A
* β-mercaptoethanol – BioRad catalog number 161-0710
* 70% ethanol – prepare this with RNase-free water and molecular biology grade ethanol
* Homogenizers – Ambion catalog number 12183-026
* PureLink DNase – Ambion catalog number 12185010
#### Procedure
* Transfer the homogenate-supernatant to a homogenizer filter-column.
* **Centrifuge** for 2 min at 12,000 ×g. The flow-through contains the RNA.
* If the filter clogs, not allowing most of the liquid through, you can pipette it up and down to dislodge the sediment and spin it again at a higher speed. Alternatively, you can move this fluid to a new homogenizer, and combine the flow-through from both.
* **Add one volume of 70% ethanol** and mix well by shaking or vortexing. Be careful—the lids of the homogenizers’ collection tubes don’t stay closed on their own.
* **Transfer up to 700 μl of sample to a spin cartridge**. If you have more than 700 μl, you’ll repeat this step until the entire sample has been passed through the spin cartridge.
* **Centrifuge** for 30 seconds at 12,000 ×g. The cartridge matrix will bind the RNA. Discard the flow-through.
* Repeat the last two steps until all the sample has been passed through the spin cartridge.
* This is a good time to prepare the DNase enzyme mix (see below).
* Add 350 μl of **Wash Buffer I** to the spin cartridge.
* **Centrifuge** for 30 seconds at 12,000 ×g. **Discard the flow-through and the collection tube**. Place the spin cartridge in a new collection tube.
* Add 80 μl PureLink DNase mix directly onto the matrix of the cartridge. Incubate at room temperature for 15 minutes.
* To prepare the PureLink DNase solution. Mix the following reagents together in a separate tube. Scale this recipe up depending on the number of spin cartridges. Also note that this enzyme mix can be prepared at any time before it’s needed.

| Reagent | Volume |
| --------------------------- | ------ |
| 10X DNase I reaction buffer | 8 μl |
| resuspended DNase |10 μl |
| water (RNase-free) | 62 μl |
| total volume | 80 μl |

* Add 350 μl of **Wash Buffer I** to the spin cartridge.
* **Centrifuge** for 30 seconds at 12,000 ×g. **Discard the flow-through and the collection tube**. Place the spin cartridge in a new collection tube.
* Add 500 μl of **Wash Buffer II** to the spin cartridge. Be sure this buffer has had ethanol added.
* **Centrifuge** for 30 seconds at 12,000 ×g. Discard the flow-through and reuse the collection tube.
* Add 500 μl of **Wash Buffer II** to the spin cartridge. Be sure this buffer has had ethanol added.
* **Centrifuge** for 30 seconds at 12,000 ×g. Discard the flow-through and reuse the collection tube.
* This is a good time to start labeling your recovery tubes.
* **Centrifuge** for 1 minute at 12,000 ×g to dry the matrix. Discard the collection tube. Place the spin cartridge into a recovery tube (or a regular RNase-free 1.5 ml microfuge tube).
* Add 30 μl of **water** (RNase-free) to the matrix.
* Wait 1 minute for the water to permeate the matrix.
* **Centrifuge** for 1 minute at 12,000 ×g.
* Pipette the elutant from the recovery tube back onto the same cartridge. Wait 1 minute, then spin again. This will insure maximal yield.
* Discard the spin cartridge.
* You now have total RNA!
* **Label** your “total RNA” with the species, population, sex, genotype, and/or tissue of origin and your initials and today’s date.
* Use the NanoDrop spec to **measure the concentration** of the total RNA. Record this information in your notebook and on the side of the tube. Also note the purity of the sample. Pure RNA should have an A260/A280 ratio > 2.0
* **Store RNA at -80˚C** and plan to use it as soon as possible.
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### RNA isolation using the Maxwell 16 automated purifier
#### Materials
* Maxwell 16 LEV simplyRNA Tissue Kit – Promega catalog number AS1280
* Heat block set to 70˚C.
#### Procedure
* Turn on the Maxwell.
* Verify that the instrument settings indicate an “**LEV**” hardware configuration and “**Rsch**” operational mode. If it's not, use the set-up menu to switch from SEV to LEV and replace the hardware (the magnetic rods and sample tray).
* For each RNA extraction, **place a cartridge in the LEV rack** with the label side facing away from the Elution Tubes. Press down on the cartridge to snap it into position.
* If you are processing fewer than 16 samples, center the cartridges on the rack.
* Carefully **peel back the seal** so that all plastic comes off the top of the cartridge.
* **Place an LEV Plunger in well 8** of each cartridge. Well 8 is closest to the Elution Tube.
* Place labeled, 0.5 ml **Elution Tubes in the front** of the LEV Cartridge Rack.
* **Label** tubes “total RNA” with the species, population, sex, genotype, and/or tissue of origin and your initials and today’s date.
* **Add 50 μl of nuclease-free water to the bottom of each Elution Tube**. For more concentrated eluate, use as little as 30 μl of water, however this may reduce total RNA yield.
* Heat the homogenate-supernatant (no more than 200 μl per cartridge) to **70˚C for 2 minutes**.
* Allow the samples to **cool for 1 minute** at room temperature.
* **Add 200μl of lysis buffer** per 200 μl of homogenate-supernatant.
* **Vortex** for 15 seconds.
* **Transfer** all 400 μl of lysate **to well 1 of the Maxwell LEV cartridge**.
(Well #1 is closest to the cartridge label and farthest from the elution tube)
* **Add 10 μl of DNase to well 4** (yellow). For less than 5 mg tissue, use only 5 μl of DNase.
After adding the blue DNase to the yellow reagent, the well should be green.
* Select **Run** on the Menu screen, and press the Run/Stop button to select the method.
* Select **RNA**, select **simplyRNA**, then select **simplyRNA once more** on the Menu screen. Next select OK at the Verification screen.
* **Open the door** when prompted. Press the Run/Stop button to **extend the platform**.
* Transfer cartridges to the instrument platform.
* **Verify** that the cartridge rack is level on the instrument platform, samples were added to well 1, cartridges in the rack are loaded on the instrument, Elution Tubes are present with 50μl of nuclease-free water and LEV Plungers are in well 8. Well 4 should be green to indicate that DNase was added.
* **Press the Run/Stop button**. The platform will retract.
* **Close the door**. Maxwell will then isolate the RNA!
Watch the timer to see when it will be done.
* **When the run ends**, the screen will prompt you to **press the Run/Stop button** to extend the platform.
* Remove the LEV Cartridge Rack from the instrument.
* Remove Elution Tubes containing total RNA and close the tubes.
* If paramagnetic particles are present in the elution tubes, centrifuge at 10,000 ×g for 2 minutes. Transfer the supernatant to a clean tube.
* Remove and discard the cartridges and plungers from the LEV Cartridge Rack.
* Use the NanoDrop spec to **measure the concentration** of the total RNA. Record this information in your notebook and on the side of the tube. Also note the purity of the sample. Pure RNA should have an A260/A280 ratio > 2.0
* **Store RNA at -80˚C** and plan to use it as soon as possible.
---
### cDNA synthesis
Isolation of total RNA from tissue is the first step towards measuring gene expression or cloning new gene sequences. The next step will be to make complementary DNA (cDNA) using RNA as a template. This is a simple procedure and there are many kits that provide reverse transcriptase and primers for this purpose. For many years, we have used the Bio-Rad iScript Select cDNA Synthesis Kit (#170-8897). This kit can be used in the same way for cDNA synthesis, whether the intended application is qPCR or less sensitive, standard PCR. If you’re preparing cDNA for qPCR use extra caution in your pipetting. If you make a master mix to make cDNA from several RNA samples at once, be sure you don’t short-change the volume of the last reaction. Also think carefully about the design of your experiment, so that you can prepare most (or ideally all) templates in parallel. For RACE-ready cDNA, use the "RACE(dT)20" primer, which adds the GeneRacer 3' primer and nested primer binding sites.

#### Be sure you're using the right reagents!
Be sure you're using the "BioRad iScript Select cDNA Synthesis Kit" reaction mix (catalog number 170-8896 or 170-8897). This one allows you to add your own primer. A kit with the annoyingly close name, " BioRad iScript cDNA Synthesis Kit" (catalog number 170-8890 or 170-8891) has a mixture of random hexamers and oligo-dT primer already in its reaction mix. While this might seem like a nice time saver, in fact priming by random hexamers is not ideal for realtime PCR.

#### Procedure (modified from the iScript Select cDNA manual)
* **Thaw** components and ensure they are well mixed. (However, keep the reverse transcriptase at -20˚C until it is added to the reaction.)
* If you haven't already done so, **measure the concentration of your total RNA** sample using the NanoDrop spectrophotometer. Calculate the volume needed for 1 μg of total RNA (x).
* For each **cDNA synthesis reaction**, mix the following 20 μl reaction in a 200-μl PCR tube. Add enough water to make the total reaction volume 20 μl.

| Reagent | Volume |
| --------------------- | ----------- |
|water (nuclease-free)| (14 – x) μl|
|oligo-dT20 primer 10 μΜ | 1 μl |
|RNA sample (1 μg) | x μl |
|5X iScript Select reaction mix|4 μl|
|iScript reverse transcriptase| 1 μl|
* **Mix** by pipetting up and down.
* **Incubate** at 42˚C for 90 minutes, then 85˚C for 5 minutes (to denature the RT).
This is stored as the thermocycler program “iscript rt”.
* Store the product at -20˚C.
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For qPCR, it is recommended that a single 50-μl reaction use 2 μl of cDNA prepared this way. For standard PCR, the cDNA may be diluted by the addition of 80 μl 1X TE buffer (to 100 μl total volume).
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