RNA extraction protocol

Protocol for: RNA extraction protocol
Prepared by: Rong Yuan
Reviewed by: David Harrison
Last revision date: 05/01/2006

RNA extraction protocol

Rev. 8/06/04

*Catalog numbers for all reagents are listed at the end of this protocol.

Exceptions:

For Reticulocytes, spin sample for 5 minutes @ 1800rpm, then draw off the

RNAlaterÒ and add TRIzolÒ Reagent to sample.

For cell culture: spin samples for 15 minutes @ 2000rpm, then draw off RNAlater and

add TRIzol Reagent to sample.

NOTE: Do not pour off RNAlater! Pellets are not tightly packed; therefore, it is possible

to lose cells.

Initial preparation (Always wear gloves)

1. Thaw tissues.

2. Fill out extraction log sheet located on \\Wonderland\G\GES Common Files\GES

Templates\Extraction Log sheet.xls.

3. Use RNaseZAPÒ to clean all tools, pipettes, bench and scale area before starting.

4. Obtain weigh boats.

5. Set up appropriate number of tubes needed for weighing (1.5ml tubes) and

homogenizing (14ml tubes if £ 4ml TRIzol, 50ml conical tubes if > 4ml TRIzol).

6. Pour RNAlater into a 14ml tube (enough for 500ml per 1.5ml tube and 2ml per weigh

boat).

7. Aliquot 500ml RNAlater into each 1.5ml tube.

8. Prepare cleaning tubes for the homogenizer: 15-20ml RNase Away and “Gibco”

dH 2 O in 50ml conical tubes.

9. Make sure correct size probe for the Polytron is hooked up (use large probe for 3 2ml

TRIzol).

Weighing tissue

1. Zero the balance with about 2ml RNAlater in a weigh boat.

2. Use a pair of forceps to pick the sample out of the tube. Tap the sample [several

times] on the side of the tube to rid tissue of excess RNAlater.

3. Place the sample in the weigh boat with RNAlater in it and record the amount of

tissue on the extraction log. Place tissue back in the original tube if extracting the

whole sample or a new tube with RNAlater if extracting a portion of the sample.

4. If only extracting a portion of the original sample, measure remaining tissue as

described above and record on extraction log. Store excess tissue @ -80°C

5. Clean forceps with RNaseZAP and use new weigh boats between each sample.

Preparation for homogenization

Always wear lab coat, gloves and eye protection when using TRIzol Ò Reagent.

1. Pipet amount of TRIzol needed into a 50ml conical tube in the hood. 1ml of TRIzol is

needed for 50-100mg tissue for most samples. Liver and tumor samples need about

5ml TRIzol per 100mg tissue.

2. Aliquot TRIzol into individual tubes with a pipet, leaving 15-20ml in the 50ml

conical for rinsing probe.

3. Dispose of pipet in pipet box in hood.

Homogenizing tissue

Notes:

* When using > 4ml TRIzol, homogenize in 50ml conical tube.

* Transfer sample to 14ml tube before spinning.

* Never operate Polytron without liquid.

* Setting for Polytron should be @ 6.

1. Cleaning probe

a. Rinse probe with RNase Away for 10-20 seconds.

b. Rinse probe with “Gibco” dH 2 O for 10-20 seconds.

c. Rinse probe with TRIzol for 10-20 seconds.

2. Homogenizing sample

a. Use forceps to pick sample out of 1.5ml tube and put in the corresponding

TRIzol tube (do not touch TRIzol with the forceps because tissue may become

sticky and difficult to remove from forceps).

b. Place forceps in RNase Away tube between samples.

c. Homogenize sample.

i. £ 5ml TRIzol, homogenize for 10 seconds, repeat as needed.

ii. 5-15ml TRIzol, homogenize for 15 seconds, repeat as needed.

iii. 15-20ml TRIzol, homogenize for 20 seconds, repeat as needed.

d. Check tube to see if the sample has been completely homogenized (should not

be able to see pieces of tissue).

e. Check the probe for pieces of tissue before homogenizing next sample.

f. Rinse probe 10-20 seconds between samples.

i. Same strain and tissue, rinse with TRIzol.

ii. Different strain or tissue.

1. Rinse with TRIzol.

2. Rinse with RNase Away.

3. Rinse with “Gibco” dH 2 O.

4. Rinse with TRIzol.

g. Continue as above homogenizing rest of samples.

3. Clean-up procedure

a. Rinse with TRIzol, RNase Away, and “Gibco” dH 2 O for 10-20 seconds each.

b. Shut homogenizer off (green button).

c. TRIzol, RNase Away, and dH 2 O waste goes into the phenol waste container in

the hood.

d. RNAlater waste can go down the sink running the water for 10 minutes.

e. Make sure bench paper in the hood is clean of TRIzol. If not, dispose of

immediately and replace with new bench paper.

4. Options for homogenized samples

a. To process samples immediately, continue following protocol below.

b. Samples can be stored @ -80°C if they cannot be processed immediately.

Extracting RNA

Note : Turn centrifuge (Sorvall RC-5B Refrigerated Superspeed Centrifuge) on and spin empty

rotor 10’ to bring to temperature. (SS-34 rotor holds 8 samples, SA-600 rotor holds 12 samples).

(Centrifuge speeds will vary with regards to the type of rotor being used: the SS-34 should be

spun at 10K and the SA-600 should be spun at 9K to avoid increased G forces which may break

tubes).

1. Incubate homogenate @ RT for 5 minutes. If samples were previously frozen, start

spin immediately after thawing.

2. Centrifuge 10K for 15 minutes @ 4°C to spin out debris.

a. While spinning, set up and label 14ml tubes for next step.

b. Place any tubes or tips that are used for phenol or chloroform in a plastic bag

and tie off bag before disposing in burn bin.

3. Transfer supernatant to a new 14ml tube, pouring away from the pellet, which is

cellular debris and DNA.

a. If there is an oily/fat layer on top, pull off fatty layer with a pipetman then

pour supernatant into new tube.

b. If the pellet moves, pull off liquid with a pipet.

4. Add 0.2ml chloroform per 1ml TRIzol to each tube and close tubes.

5. Shake tubes vigorously by hand for 15 seconds.

6. Incubate samples @ RT for 10 minutes.

7. Centrifuge 10K for 15 minutes @ 4°C.

a. Label more 14ml tubes for next step.

8. Transfer aqueous phase (top layer) into a new tube. Avoid interface so as to not

contaminate RNA with DNA and proteins.

9. Add 0.25ml high-salt buffer (recipe included at end of protocol) per 1ml of TRIzol

to each sample.

10. Add 0.25ml isopropanol (isopropyl alcohol) per 1ml TRIzol to each sample.

11. Mix well by gently inverting tubes 3-4 times.

12. Incubate @ RT for 20-30 minutes.

13. Centrifuge 10K for 15 minutes @ 4°C.

a. During spin, obtain ice, a 1.5ml tube of “Ambion” dH 2 O, and 1.5ml tubes.

b. Place dH 2 O and labeled tubes on ice.

14. When spin is done, place samples on ice (from this point forward always keep

samples on ice).

15. Pour liquid (high salt/isopropanol) into a liquid waste container leaving the pellet

behind. Tap tube on a paper towel to get as much liquid out as possible and place

tube back on ice.

16. Add 5ml cold 75% ETOH to each sample to wash the pellet.

17. Pour off 75% ETOH into liquid waste container leaving between 0.5-1.0ml with the

pellet.

18. Transfer the pellet and 75% ETOH to a 1.5ml tube by pouring contents of 14ml tube

into the 1.5ml tube. If pellet does not transfer to the 1.5ml tube, pour 75% ETOH

back into 14ml tube and repeat process until pellet transfers.

19. Pull off residual 75% ETOH with P1000.

20. Add 1ml 75% ETOH to each tube.

21. Vortex pellet gently to remove residual high salt/isopropanol (it is critical to remove

as much as possible because it will show up in Agilent Bioanalyzer chromatogram as

a “hump” under the 28S peak and may skew ratio measurement). If pellet breaks

apart, spin at no more than 9,000 rpm (7,500g) for 5 minutes @ 4°C (walk-in).

22. Remove residual 75% ETOH and high salt/isopropanol residue with P1000.

23. Quick spin and remove remaining 75% ETOH with P100. Repeat process with P10

until no residual 75% ETOH remains.

24. Air dry pellet for 10-30 minutes on ice w/ KimWipe on top of the open tubes. If

pellets are thick, drying time may increase. Dry to completion as residual ethanol can

inhibit downstream applications.

25. Dissolve pellets using “Ambion” dH 2 O.

a. Amount of water depends on size of the pellet.

b. Add appropriate amount of water to achieve a concentration of 2-7mg/ml.

26. Leave pellets on ice for 10 minutes. Vortex occasionally.

27. Incubate samples @ 60°C for 10 minutes, vortexing after 5 minutes.

a. Make sure the pellets are resuspended.

28. Vortex and place tubes back on ice for 1 minute.

29. Spin @ 4°C (walk-in) for 5 minutes @ max speed to remove insoluble material.

30. Transfer supernatant to a final 1.5ml tube.

31. Check quantity by O.D. (A260/280) using the NanoDrop.

32. Write final concentration and date on side of tube before storing them.

33. Check quality using the Agilent Bioanalyzer 2100.

34. Fill out the rest of the extraction log sheet.

Guidelines for splitting and storage of RNA

-Split RNA into aliquots with no more than 300ml per tube.

-Storage of RNA

If RNA will be used within 1 month, store in H 2 O @ -20°C.

If RNA will not be used for 2 months or longer, precipitate it and store @ -80°C.

Reagents

RNAlaterÒ (Ambion Incorporated, Cat# 7021)

TRIzolÒ Reagent (Invitrogen Life Technologies, Cat# 15596-018)

RNaseZAPÒ (Ambion Incorporated, Cat# 9780)

RNase Away* Surface Decontaminant (Fisher Scientific, Cat# 14-375-35)

“Gibco” Ultrapure DNase/RNase-Free Distilled Water (Invitrogen Life Technologies,

Cat# 10977-015)

“Ambion” Nuclease-free Water (Ambion Incorporated, Cat# 9930)

Isopropyl Alcohol (Amresco Incorporated, Cat# 918)

High-salt buffer (0.8M NaCitrate/1.2M NaCl)

23.5g NaCitrate

24ml 5M NaCl

“Gibco” Nuclease-free Water (V f = 100ml)