RNA isolation

1

 Thaw the lysates on ice!

2

Put gloves on. Spray your bench 2x with RNase-Away / 2% SDS and clean the work surface with a tissue.

3

Work in the fume hood.

• Pipette 0.2 * V_GTC BCP into the samples.
• Vortex briefly until a white-yellow cloudy solution forms.
• Incubate 5 min (RT)

4

Centrifuge the samples for 15 minutes at a speed of 12,000x g at 4 ^oC.

5

Very carefully transfer the top (aqueous) phase to a new RNase-free eppendorf tube. You continue with this eppendorf tube.

Watch the video in the interactive potocol again for instructions.

6

• ·Add RNase-free isopropanol to the samples (Ratio 1: 1)
• Homogenize the samples by gently inverting the eppendorf tubes a few times. 
• Incubate 10 minutes (RT)

7

Centrifuge the samples for 30 minutes at a speed of 12,000 x G at 4 ^oC.

8

Discard the supernatant. You can pat the eps upside down against a clean tissue to get the last bit of liquid out of the eppendrof tubes.

9

Add 100 µL RNase-free 75% EtOH to the samples. Vortex the samples briefly.

10

Centrifuge the samples at 15,000 x G for 5 minutes at 4^oC.

11

• Gently remove the supernatant by pouring off the supernatant in one gentle motion.
• If necessary, remove large drops of supernatant, on the side of the eppendorf tube, by pipetting with an RNAse free pipette tip without touching the pellet.
• Put the eppendorf tube upside down on a clean tissue to air dry for 5-10 minutes.

12

You can work on the table from now on. 

Dissolve the samples in 20 µL RNase-free MilliQ.

13

The RNA concentrations of the samples are measured with the DS-11 spectrophotometer.

As soon as 2-4 teams have finished their RNA isolation, an assistant will measure it for you.

Which components does the supernatant of the RNA precipitation contain?

1. DNA
   Correct = no
2. Salts en isopropanol
   Correct = yes
3. Protein
   Correct = no
4. Cell components
   Correct = no

Feedback if incorrect: Think about the steps of the RNA isolation process. Which components are needed in the supernatant and which components need to be discarded?

Select which operations are not required to maximize your RNA yield

• Which things do we consider as “clean” and which are “dirty”?

1. Keeping your eppendorfs on ice
   Correct = no
2. Spray your hands with RNase-away every time you touch something that might have RNA on it
   Correct = yes
3. Keeping eppendorfs closed where possible
   Correct = no
4. Working inside the fumehood
   Correct = yes
5. Using RNA-free pipet tips
   Correct = no
6. Leaning over closely to see if there is still some solution stuck in a vial or Eppendorf
   Correct = yes
7. Pressing the eppendorfs deep into the ice to keep them cold
   Correct = yes
8. Touching your face during the experiment
   Correct = yes

Feedback if correct: Correct! Keep in mind that working in a fumehood does not change the amount of RNase. However this does not mean that you shouldn’t work in a fumehood!! You might use some hazardous chemicals in the protocol that are only to be used inside the fumehood.

Ice is generally “dirty”. You don’t want to push the eppendorfs too deep into the ice, because they’ll get contaminated on the sides. You’ll definitely touch them to open the top and worse, you’ll probably touch the ice with your “clean” gloves.

Your skin (and therefore your face) is full of RNase! Do not touch your face during the experiment. For a similar reason, don’t lean in closely to check something on your setup. It happens often that a hair (or eyelash, etc) end up in your experiment. Especially when working with PCR plates it is important to keep everything clean.

Feedback if incorrect: Think of what is really necessary to work RNase-free. Which operations do we do for different reasons?

The phase partition step in RNA isolation is often described using chloroform. For what reason is bromochloropropane (BCP) often used as an alternative for this step?

a.     BCP is used instead of chloroform because it is less toxic.
Correct = yes

b.     Synthetic production of BCP is less complex and cheaper than chloroform.
Correct = no

c.      Chloroform is very susceptible to degradation over time, whereas BCP is not.
Correct = yes

d.     The use of BCP decreases the possibility of contaminating RNA with DNA.
Correct = yes

e.     Chloroform is colourless, whereas BCP is not. The phase partition is hard to observe with a colorless liquid.
Correct = no

f.       Generally speaking, BCP is most suitable for human tissue, whereas chloroform is more often used for other mammalian tissues.
Correct = no

Feedback if correct: That is correct.

Feedback if b: Synthetic production of BCP involves many steps and reacting agents and is in general terms more expensive to obtain compared to chloroform.

Feedback if e: Both BCP and chloroform are colorless.

Feedback if f: BCP usage does not hold restrictions on human tissue and is also commonly used for mammalian tissue.

What should you do when there is two phases in your pipette tip after attempting to phase-separate the water phase from the organic phase?

a.     Repeat the centrifugation step.

1. • Correct! By centrifuging the sample a clear separation is made again between the water phase and the organic phase – this separation might have been disturbed. It is easier to work with clearly separated phases as you can distinguish them by eye and place your pipet on the correct place.

b.     Put the water phase back into the Eppendorf and pipet off the water phase again.

1. • Incorrect. You might disturb the clear separation of the phases by putting the water phase back into the Eppendorf, consequently making it more difficult for yourself with pipetting off the water phase again.

c.      Taking some organic phase out together with the water phase is not a problem.

1. • Incorrect. Taking some organic phase out together with the water phase will make the first separation contaminated with DNA and/or protein.

d.     You need to start all over with the RNA isolation from the first step of the protocol.

1. • Incorrect. Both the water phase and organic phase are still present and only a correct separation is needed to solve this problem.

How should the Eppendorf look after correct separation?

1. 
   Incorrect. This picture shows mixed phases.
2. 
   Incorrect! This is how the phases look after centrifuging.
3. 
   Correct! This is how the water phase looks after correctly separating it from the organic layer.
4. 
   Incorrect. Here you can clearly see that there is organic phase in the supposedly clean water phase.