RNA extraction with TriZol - KravitzLab/KreedLabWiki GitHub Wiki

Material

  • Plastic Potters (Fisherbrand™ RNase-Free Disposable Pellet Pestles)
  • P1000/P200/P20
  • Pipettes tips with Filters
  • Eppendorf (1.5mL) tubes RNAse free
  • TriZol
  • Chloroform 100%
  • Isopropanol
  • Ethanol 75% (At -20 °c)
  • RNAse free water
  • Centrifuge
  • Nanovue/Nanodrops
  • Box of gloves -Fume hood
  • Containers for used TriZol
  • Water bath with heater
  • Dry Ice
  • Ice
  • Box of gloves
  • RNA grade Glycogen

Setting up the area for RNA extraction to avoid RNA degradation and for safety

  1. Always work on the Fume hood the first day because of the TriZol and chloform usage.
  2. Spray every surface and pipettes and boxes and areas of contacts with RNAse Away/Anti RNAse solutions. If you do not have any, you can use a bleach solution diluted to 1%.

Unpurified RNA such as in brain punches is unstable at room temperature because of RNAse activity. It takes around 10-30 minutes for a RNAse to destroy RNA at room temperature, therefore cleaning the area with anti RNAse solution is helpful to avoid potential RNA degradation

  1. Work with a lab and gloves to avoid RNAse contact
  2. Have a beaker with purified water/RNAse free water to clean the plastic potters

Set up in the fume hood

Protip for RNAse extraction to increase the yield

  1. Before doing any lysis with TriZol, you must keep the samples in dry Ice at -60 to -80. RNAse activity is high at room temperature, slow at 4°c, almost inhibited at -20°c, and completely blocked at -80°c.

2) Never hesitates to change gloves and spray some more anti RNAse solution on the surfaces and on your gloves

  1. Keep the pipettes boxes closed as much as possible
  2. If you have a large cohort (more than 15 samples), split it in several groups. Do 10 in the morning and 10 in the afternoon.
  3. Since TriZol denaturates RNAse, once you put TriZol in your eppendorf with your punch, your sample is "safe", you can leave it on ice or at room temperature without any issues after lysing the tissue.
  4. Use RNA grade glycogen (5-20ug) : Glycogen helps carryover the nucleic acid during the precipitation phase with isopropanol
  5. AVOID transfering the punch in another eppendorf. Try to do the extraction in the tubes where you originally put the punch in.
  6. Discard any pipette tips that touches the edges of a tube.

RNA extraction step by step : First Day

1- Add 800uL of Trizol to the eppendorf with the brain punch (Change tips each time unless you do not touch the edge of the tubes !!!)

For big punches such as the PFC you can add 1mL of Trizol instead of 800uL

2- With the plastic potter (new or cleaned) do some ups and downs to lyse the punch. Do it throughly but slowly to avoid spilling the TriZol out of the tube. This step should take 1-2 minutes for small samples (Ex: VTA) and 5 minutes for big structures (Ex : PFC). It should be finished when you do not see the brain punches anymore like it has "disolved" in the TriZol. Do ups and downs and try to "smash" the structure on the side of the tube.

3- Once you are finished with a tube, put in on normal ice.

4- Between each samples CLEAN THE PLASTIC POTTER IN ULTRAPURE WATER (or use a new one).

5- Once each sample is homogenized in TriZol, let it sit at room temperature for 10 minutes so TriZOl can dissociate the nucleic acids.

6- During this time, set the Centrifuge to 4°c (Fast Temp).

7- After 10 minutes, add 160 uL of chloroform in the tubes (If you added 1mL of TriZol, put 200uL).

8- Vigorously shake the tubes for 15 seconds to allow for good homogeneisation

9- Let the tubes sit at room temperature for 2-3 minutes.

10- Centrifuge the tubes at 4°c 12 000 RPM for 10 minutes

11- Remove the upper layer part of the tube (See the schematic) : It should be around 200uL, take as much as you can without disturbing the interphase, if you disturb the interphase, just centrifuge the tube for 2 minutes again, put the recovered upper layer in another eppendorf tube.

NB : When the interphase is quite visible (white layer, more or less thick), it usually mean that your extraction is successfull because it means that you had quite a lot of biological material to begin with, do not freak out if the interphase is not super thick though, it's not unusual when dealing with very small samples

11-B OPTIONNAL : Add 1uL of RNA grade glycogen 20mg/mL to add 20ug of glycogen in the separated phase. It's optionnal because glycogen helps carryover the nucleic acid and can help increase the yield, if you are dealing with rather big/RNA rich structures, you might not need to add it.

12- Add 400 uL of Isopropanol to the new eppendorf tube with the aquaeous phase, then mix vigorously for 15 seconds.

13- Incubates the samples overnight at -20°c

END OF THE FIRST DAY

RNA extraction step by step :Second day

1- Set the centrifuge at 4°c (Fast temp)

2- Recover the tubes from the -20°c freezer after the overnight precipitation

3- Centrifuge them (12 000 RPM, 4°c) for 15 minutes

4- After centrifugation, throw away the content of the tubes in the trash (Yes, throw it out ! we only cares about the RNA pellets that is at the bottom)

5- Take the Ethanol 75% solution from the freezer, keep it in dry ice to keep it cool

6- Take 1mL of Ethanol 75% and GENTLY push it in and out in the tubes to unstick the RNA pellets at the bottom of the tubes. BE EXTREMELY CAREFUL TO NOT ASPIRATE THE PELLET. Do it gently a few times, this step is crucial to clean out the RNA !

When you have quite a lot of RNA or when you use a carryover such as glycogen, the pellet can be visible, it looks like a little white feather

7- Centrifuge the tubes 13000 RPM at 4°c for 2 minutes.

8- Empty the tubes once again in the trash after centrifugation

9- Proceed with another EtOH 75% Wash.

10- Centrifuge the tubes 13000 RPM at 4°c for 2 minutes

11- Empty the tubes once more

12- Dry out the tubes by letting them rest flat down on a paper towel for 15 minutes. It's important to not overdry the pellets or else it will be hard to resuspend it.

13- Add 20uL of RNA-free water in the tubes (do aspirations and reflux to solubilise the RNA)

NB : When you have a LOT of RNA (which can be the case of rats PFC or DS, then eluting in 30uL is more advised)

14- Incube the RNA tubes for 5 minutes at 60°c in a water bath.

15- Once it's finished, the RNA should be kept at -80°c until utilisation

Measuring RNA concentration and controlling RNA quality

RNA concentration and quality is measured with the Nanodrop machine, it gives you a concentration in ug/ml and the 260/280 ratio and 260/230 ratio. Those ratio measure "What is absorbing at 260 nm / what is absorbing at 280 nm or 230 nm". These ratios can testify of your RNA quality and should be around 1.8-2.0.

1- Press the screen of the Nanovue to start

2- Press on "RNA"

3- Clean the pedestal with 2uL of RNAse free water

4- Load 2uL of RNAse free water and press on "Blank" to set the values to 0

5- Load your sample (2uL, change tips each time !!!!) and press on "Measure"

6- Write down the values you obtain

Common issues/questions and their solutions/answers

My RNA concentration are pretty low :(

Depending on what you are using the RNA for, this can be a non issue. It can be difficult to obtain a good RNA concentration for small brain structures. For qPCR purposes, 100ng of RNA in total is enough to run an effective reverse transcriptase and for making the PCRs, especially if you are looking at well expressed genes. Ideally, you'd aim to run a reverse transcriptase with 500 to 1000 ng of RNA.

For future experiments, going faster with the lysis, RNAse away cleaning and recovering more of the upper layer after adding chloroform can help increase the yield. Adding 20ng of glycogen can help increase the yield as well.

My 260/280 ratio is below 1.8 :(

It should be fine. Your RNA isn't totally pure, a "low" 260/280 (1.5-1.8) is a sign that you might have some phenol contamination, which can happen with TriZol extraction, but it shouldn't affect your qPCR results.

For future experiments, using filtered tips, making sure to not disturb the interphase when recuperating the upper layer with RNA can help increase the cleanliness of RNA. You can also let the pellet dry a little bit longer, make sure all drops of Ethanol are gone, or even add another cleaning steps with some ethanol 95%. If your ratio is below 1.5, I wouldn't use the sample, the contaminant could impact your results...

My 260/230 ratio is below 1.8 :(

This ratio is less problematic, especially with TriZol extraction. In MOST of the cases, the low 260/230 ratio is a sign of carrying over some TriZol salts, especially guanidinium thiocyanate that absorbs at 230 nm. Previous work (https://pmc.ncbi.nlm.nih.gov/articles/PMC5944930/) have shown that this ratio is not crucial for qPCR amplification. It's better to have a high ratio (adding another ethanol cleaning step).

If you are working with a low RNA concentration (less than 20 ng/ul), then having a low 260/230 ratio is almost normal. If your ratio is REALLY low (less than 0.5), you can still run the qPCR but look at the amplification of the control gene.

OMG OMG I LET MY RNA AT ROOM TEMPERATURE FOR A FEW HOURS !!!

Ok so it depends at which steps you are on.

  • Is it the brain punch that you forgot outside of the -80 freezer ? yeah the sample is cooked :(. If it's less than 30 minutes, it's still worth a try !
  • It's the sample lysed in TriZol ? It should be fine. I'd be more cautious for the next steps of extraction and look at the yields and results of PCR. TriZol should inhibits any RNAse activity.
  • It's the purified RNA in RNAse free water ? It's fine. Theorically at this step, RNA should be pure so it should be pretty stable, even at room temperature. I would still let them sit at MAXIMUM 4°c and not room temperature though. RNA should be stored at -80 degrees celcius to prevent any degradation just in case.

Why should I do TriZol extraction and not kit ?

You should do whatever you feel more comfortable with to be honest. RNA extraction kit work very well, TriZol extraction also works very well. Both have their strength and weaknesses.

The kits strengths:

  • Take less time
  • The RNA quality is higher
  • Less teps

The kits Weaknesses:

  • The RNA yield is usually a bit lower which can be a problem for small samples(See De Sa Nogueira David PhD thesis 'Voluntary cocaine or sugar intake induce neuroadaptations of the endocannabinoid system in reward-related brain regions', 2019) which can be a problem for small samples

Trizol Extraction strenghts :

  • Much less expensive
  • Can help for isolating RNA, DNA and proteins
  • Enhanced RNA yields

Trizol extraction weaknesses :

  • Lower RNA quality
  • Longer protocols
  • Work under the fumehoods with irritant products