FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.

RNA Extraction from

Mammalian Tissues

Reagents
Choose the most appropriate kit for your sample. Consult the Qiagen web site for more
specifics, or call Qiagen technical support (1 (800) 362-7737):
• Qiagen RNeasy® Mini Kit (12): Qiagen (Cat. No. 74104). Yields <100 μg RNA from
0.5 - 30 mg tissue or 1x105 to 1x107 cells.
• Qiagen RNeasy® Midi Kit (12): Qiagen (Cat. No. 75142). Yields <1 mg RNA from 20 -
250 mg tissue or 5x106 - 1x108 cells.
• Qiagen RNeasy® Maxi Kit (12): Qiagen (Cat. No. 75162). Yields <6 mg RNA from
150 mg – 1 g of tissue or 5 x 107 – 5 x 108 cells.
TRIzol® Reagent (a ready to use mixture of phenol, guanidine isothiocyanate, red dye and other
proprietary components): Invitrogen (Cat. No. 15596-026)
TRIzol® LS Reagent (recommended for liquid samples e.g. FACS sorted cells): Invitrogen
(Cat. No. 10296-010)
Chloroform: Sigma (Cat. No. C-2432)
RNaseZap® RNase Decontamination Solution, 250 ml: Ambion (Cat. No. 9780)
RNase-free Water: Ambion (Cat. No. 9932 or 9922)
100 % (200 proof) Ethanol: Pharmco (Cat. No. 111ACS200)
70% Ethanol (in RNase-free H2O)
50 ml Falcon Tubes (BD Cat. No. 352070). 9400 RCF rating. Required for Maxi Kit.
15 ml Falcon Tubes (BD Cat .No. 352097). 6000 RCF rating. Required for Midi Kit.
1.7 ml Microcentrifuge Tubes (Denville Scientific, Cat. No. C-2170). Required for Mini Kit.
Agilent 2100 bioanalyzer: (Agilent Technologies, Cat. No. G2940CA)
Agilent RNA 6000 Nano Kit: (Agilent Technologies, Cat. No. 5067-1511). For RNA in the
concentration range of 25–500 ng μl-1
Agilent RNA 6000 Pico Kit: (Agilent Technologies, Cat. No. 5067-1513). For RNA in the
concentration range of 50–5000 pg μl-1

NOTE: We do not recommend the use of the RNAlater RNA stabilization reagent (this should not be confused with
Buffer RLT). Direct disruption of the tissue or cells in Buffer RLT or Trizol yields the best results in our experience.
If the tissue has been stored in RNAlater, the tissue must first be removed and placed into Buffer RLT and
immediately disrupted.
NOTE: We do not recommend DNase treatment of RNA samples for microarray analysis.
NOTE: If you are not experienced with RNA isolation, please read the literature and tips found on Ambion’s
website: [Link]

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FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.

Protocol A – Cells (<107), Islets or Small Amounts of Tissue (<30 mg)

Sample preparation and homogenization

Gloves should be worn at all times and follow standard RNA handling techniques. Lysis of the
sample in TRIzol® works well and may give higher yields due to better lysis than can be
achieved with the Buffer RLT provided in the Qiagen RNeasy kits.
a. Islets. Prepare the islets according to standard protocols, working quickly, and at the last
step remove any residual wash buffer. Resuspend the islet pellet (nor more than 500
mouse islets) in a 2.0 ml microcentrifuge tube in 1 ml TRIzol and vortex vortex well to
ensure complete lysis.
b. Cells. To a pellet of cells (<1 x 107 cells) add 1 ml TRIzol or for direct lysis of cells
grown in a monolayer, add 1 ml TRIzol (<10 cm diameter dish) and collect cell lysate
with a rubber policeman, transferring to a 2.0 ml microcentrifuge tube.
c. Sorted Cells. The issue with extracting RNA from sorted cells is the volume of cells after
sorting must not dilute the denaturing agent beyond its ability to denature RNase. As such
we recommend the use of TRIzol LS which remains active when diluted up to 1:3. If
possible, sort cells directly into 750 μl of TRIzol LS (sample volume must not exceed
250 μl – if this is a problem use more reagent and adjust the protocol accordingly).
d. Tissue. This protocol should only be followed for use with small amounts of tissue (<30
mg); if using larger amounts of tissue follow Protocol B. The volume of lysis reagent
should be at least 10 fold greater than the volume of tissue. Thus for <30 mg of tissue use
1ml TRIzol. The tissue must be completely disrupted by homogenization as detailed in
Protocol B, using a homogenizer probe that is appropriate for small sample volume in a 2
ml microcentrifuge tube. If such a probe is not available you can attempt to chop the
tissue directly in lysis reagent using very fine scissors (Fine Science Tools, Cat. No.
15012-12). Warning: incomplete lysis will reduce yield.

RNA isolation using Qiagen RNeasy® Mini Columns

1. For samples processed with TRIzol:
a. Ensure that the sample is completely lysed: if working with cells or islets vortex
well, or if working with tissue ensure complete homogenization. Samples can be
stored at this point at <-70°C for at least 1 year.
b. Incubate sample for 5 minutes in TRIzol at room temperature.
c. Add 0.2 ml of chloroform for every 1ml of TRIzol used. Shake vigorously for 15
seconds and incubate at room temperature for 2-3 min.
d. Centrifuge samples 5 min. at 12,000 x g at 4°C.
Note: The 4°C spins are essential for phase separation. Room temperature spins may result in
variable phase separation thus resulting in variable RNA yields.
e. Transfer the aqueous phase to a fresh microcentrifuge tube. Proceed immediately
to Step 2.
Note: The aqueous phase is the colorless upper phase that corresponds to ~60% of the volume of
TRIzol used. The interphase should be fairly well-defined.

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FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.

2. Add 1 volume (usually 600 μl) of 70% ethanol to the cleared lysate, and mix
immediately by pipetting. Do not centrifuge. Continue without delay with step 3.
3. Apply up to 700 µl of the sample, including any precipitate that may have formed, to
an RNeasy mini column placed in a 2 ml collection tube (supplied). Close the tube
gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-
through, but not the collection tube.
Note: If the volume exceeds 700 µl, load aliquots successively onto the RNeasy column, and centrifuge
as above. Discard the flow-through after each centrifugation step.
4. Add 700 µl Buffer RW1 to the RNeasy column. Close the tube gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the column. Discard the
flow-through and collection tube.
5. Transfer the RNeasy column into a new 2 ml collection tube (supplied). Pipet 500 µl
Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 s
at ≥8000 x g (≥10,000 rpm) to wash the column. Discard the flow-through, but not
the collection tube.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.
6. Add another 500 µl Buffer RPE to the RNeasy column. Close the tube gently, and
centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to dry the RNeasy silica-gel
membrane.
Note: Following the centrifugation, remove the RNeasy mini column from the collection tube carefully
so the column does not contact the flow-through as this will result in carryover of ethanol.
7. To elute, transfer the RNeasy column to a new 1.5 ml collection tube (supplied).
Pipet 30–50 µl RNase-free water directly onto the RNeasy silica-gel membrane.
Close the tube gently, wait 1 min, and centrifuge for 1 min at ≥8000 x g (≥10,000
rpm) to elute.
Note: Never elute with less than 30 μl water. If the expected RNA yield is >30 µg, repeat the elution
step with a second volume of RNase-free water. Elute into the same collection tube.
8. Keep eluted RNA on ice at all times and store at <-70°C.

Simplified RNA Isolation Protocol for Experienced Users

1. Complete sample pre-processing for samples processed in TRIzol:
f. Lyse / homogenize and incubate sample for 5 minutes in TRIzol at room
temperature.
g. Add 0.2 ml of chloroform for every 1ml of TRIzol used. Shake vigorously for
15 seconds and incubate at room temperature for 2-3 min.
h. Centrifuge samples 5 min. at 12,000 x g at 4°C. Transfer the aqueous phase to a
fresh microcentrifuge tube. Proceed immediately to Step 2.
2. Add 1 volume (usually 600 μl) of 70% ethanol to the cleared lysate, and mix
immediately by pipetting.
3. Apply up to 700 µl of the sample to an RNeasy mini column placed in a 2 ml
collection tube. Centrifuge for 15s and discard the flow-through. Repeat as necessary.
4. Add 700 µl Buffer RW1 to the RNeasy column. Centrifuge for 15 s as above.

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FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.

5. Transfer the RNeasy column into a new 2 ml collection tube. Pipet 500 µl Buffer
RPE onto the RNeasy column. Centrifuge as above and discard the flow-through.
Add another 500 µl Buffer RPE to the RNeasy column. Centrifuge for 2 min to dry
the RNeasy silica-gel membrane.
6. To elute, transfer the RNeasy column to a new 1.5 ml collection tube. Pipet 30–50 µl
RNase-free water directly onto the RNeasy silica-gel membrane. Wait 1 min, and
centrifuge for 1 min to elute.
7. Keep eluted RNA on ice at all times and store at <-70°C.

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FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.

Protocol B– ANIMAL TISSUES (150 to 1000 mg)

Preparation
Clean the homogenizer probe by running it at maximum speed in the probe wash tubes (50 ml
conical tubes) as follows:
I. RNAseZAP®: 30s
II. DEPC Water: 30s
III. 100% Ethanol: 30s
IV. DEPC Water: 30s

Sample preparation and homogenization

The tissue should be placed in at least 10 volumes of TRIzol® or Buffer RLT (or approximately
10 μl reagent per 1 mg tissue). Larger volumes can be used if necessary or desired. Smaller
volumes may lead to RNA degradation during processing or storage. For the purposed of this
protocol we will assume that the weight of tissue used is 1 g. If using 150-500 mg of tissue, use
half the volumes used below - consult the Qiagen RNeasy® Handbook for further details.
1. Arrange appropriately labeled 50 ml conical tubes with 15 ml of TRIzol or Buffer
RLT with 2-Mercaptoethanol in each on ice.
2. Quickly dissect out up to 150 mg to 1 g of tissue and place immediately into tubes
containing cold TRIzol or Buffer RLT on ice.
Note: If working with the pancreas, the animal should be anesthetized and the pancreas removed while the
animal is still living - immediately proceed to step 3, before processing any other samples.
3. Immediately homogenize the tissue using a conventional rotor–stator homogenizer
for at least 45 s at maximum speed until the sample is uniformly homogeneous.
4. Place homogenate on ice and when all samples are complete proceed immediately to
step 7. Alternatively, snap freeze the homogenate in liquid nitrogen and store at <-
70°C for future RNA extraction (this may not be an option with pancreas).
5. Wash the homogenizer probe as above and repeat steps 2 to 4 for each sample. When
finished, ensure that the probe is thoroughly cleaned.

RNA isolation using Qiagen RNeasy® Maxi Columns

6. If the lysate has been stored frozen, thaw quickly and transfer to a new 50 ml tube
(storage at <-70°C reduces the integrity of the tube and they should never be directly
centrifuged).
7. Dependant upon lysis method used, follow the appropriate procedure:
a. For samples processed with TRIzol:
i. Incubate sample for 5 minutes in TRIzol at room temperature.
ii. Add 3 ml chloroform (0.2 ml for every 1ml of TRIzol used). Shake
vigorously for 15 seconds and incubate at room temperature for 2-3 min.
iii. Centrifuge samples for 15 min at 10,000 x g at 4°C (9000 rpm in Sorval
SLA-600TC rotor).
Note: The 4°C spins are essential for phase separation. Room temperature spins may result
in variable phase separation thus resulting in variable RNA yields.

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FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.

iv. Transfer the aqueous phase to a fresh tube. Use only this aqueous phase
in subsequent steps and proceed immediately to Step 8.
Note: The aqueous phase is the colorless upper phase that corresponds to ~60% of the
volume of TRIzol used. The interphase should be fairly well-defined.
b. For samples processed with Buffer RLT:
i. Centrifuge the tissue lysate for 15 min at 10,000 x g at 4°C (9000 rpm in
Sorval SLA-600TC rotor). Carefully transfer the supernatant to a new 50
ml tube by pipetting. Use only this supernatant (lysate) in subsequent steps
and proceed immediately to Step 8.
Note: In most preparations a small pellet will form, sometimes accompanied by a fatty
upper layer. Transferring the pellet or the fatty layer may reduce the amount of RNA that
binds to the membrane and cause the spin column to clog. To avoid transferring
contaminants, hold the pipet tip under the fatty upper layer, and don’t disturb the pellet.
8. Add 1 volume (9 to 15 ml) of 70% ethanol to the lysate, and mix thoroughly by
shaking vigorously. Do not centrifuge. Proceed immediately to step 9.
9. Apply half of the sample, including any precipitate that may have formed, to an
RNeasy maxi column placed in a 50 ml centrifuge tube (supplied). Maximum loading
volume is 15 ml. Close the tube gently, and centrifuge for 5 min at 3000–5000 x g
(5000 rpm in Sorval SLA-600TC rotor). Discard the flow-through.
10. Repeat step 9 with the remaining sample from step 8.
11. Add 15 ml Buffer RW1 to the RNeasy column. Close the centrifuge tube gently and
centrifuge for 5 min at 3000–5000 x g to wash the column. Discard the flow-
through.
12. Add 10 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and
centrifuge for 3 min at 3000–5000 x g to wash the column. Discard the flow-through
and replace column in centrifuge tube.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.
13. Add another 10 ml Buffer RPE to the RNeasy column. Close the centrifuge tube
gently, and centrifuge for 10 min at 3000–5000 x g to dry the RNeasy silica-gel
membrane. It is important to dry the RNeasy membrane since residual ethanol may
interfere with downstream reactions.
Note: This centrifugation ensures that no ethanol is carried over during elution. Following the
centrifugation, remove the RNeasy column from the centrifuge tube carefully so the column does not
contact the flow-through as this will result in carryover of ethanol.
14. To elute, transfer the RNeasy column to a new 50 ml collection tube (supplied). Pipet
the appropriate volume of RNase-free buffer of water (0.8 to 1.2 ml) directly onto
the RNeasy silica-gel membrane. Close the tube gently. Let it stand for 1 min, and
then centrifuge for 3 min at 3000–5000 x g.
15. Repeat the elution step (step 14) as described with a second volume of RNase-free
buffer or water. To obtain a higher total RNA concentration, this second elution step
may be performed by using the first eluate (from step 14). The yield will be 15–30%
less than the yield obtained using a second volume of RNase-free water, but the final
concentration will be higher.
16. Store at -80°C.

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