Download RNA Extraction SOP

GENOMICS and SEQUENCING CENTER
RNA Extraction – Standard Operating Procedure
RNA Extraction from Bacterial Cells
Purpose:
The RNeasy Kit from Qiagen, Inc. provides a fast and simple procedure for the preparation of up to 100 µg of total RNA from
animal cells, animal tissues, bacteria and yeast (RNeasy Mini Kits) or plant cells, plant tissues and filamentous fungi (RNeasy Plant
Mini Kits). The RNeasy kits allow for simultaneous processing of samples in less than 30 minutes. All RNA molecules longer than
200 nucleotides are isolated, and the RNA can be used for standard downstream applications such as: RT-PCR, northern blotting, poly
A+ RNA selection, primer extension, RNase and S1 nuclease protection, cDNA synthesis, differential display, and expression-chip
analysis.
The following protocol outlines the steps necessary to isolate total RNA from bacterial cells, and is a modified version of the
Qiagen protocol that has been adopted by the Nelson Lab. Bacteria (maximum of 1 x 109 cells) are incubated in a buffer containing
lysozyme to digest the bacterial cell wall prior to lysis. After addition of guanidine isothiocyanate (GITC)-containing lysis buffer, the
lysate is passed through a QIAshredder column for homogenization. Then ethanol is added, and the sample is loaded onto an RNeasy
column. Total RNA binds to the RNeasy silica-gel membrane, contaminants are efficiently washed away, and high quality RNA is
eluted in RNase-free water. For information on isolating total RNA from other cells or tissues please consult the RNeasy® Mini
Handbook from Qiagen.
I. Materials:
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RNeasy Mini Kit (Qiagen #74103) – 20 RNeasy columns
QIAshredder (Qiagen #79654) – 50 columns
Ethanol (96-100%)
14.3 M β-mercaptoethanol (β-ME)
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Lysozyme
II. Determining the Correct Amount of Starting Material
The RNA content varies greatly between bacterial strains and under different growth conditions: 1x 109 bacteria correspond to
approximately 20-160 µg of total RNA. It is essential to use the correct number of bacteria in order to obtain optimal RNA yield and
purity with RNeasy columns. Two main criteria limit the maximum number of bacteria that can be used (1 x 109 bacteria):
• The RNA binding capacity of the RNeasy mini column (100 µg RNA)
• The volume of Buffer RLT required for efficient lysis.
Therefore, initial experiments should be performed with 5 x 108 bacteria. Depending on yield and purity, it may be possible to
increase the number of bacteria in subsequent preparations. Do NOT overload the RNeasy column. Overloading will significantly
reduce yield and purity.
III. Important Notes Before Starting
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If using RNeasy Mini Kit for the first time, read “Important Points before Using RNeasy Kits” (pg 16 of RNeasy® Mini
Handbook, 3rd edition).
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If working with RNA for the first time, read Appendix A (pg 88 of RNeasy® Mini Handbook, 3rd edition).
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For RNA isolation, bacteria should be harvested in log phase growth.
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Bacterial pellets can be stored at –70oC for later use, or used directly in the procedure. Frozen bacterial pellets should be
thawed slightly to allow pellets to be dislodged by flicking.
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GENOMICS and SEQUENCING CENTER
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Homogenized cell lysates (in Buffer RLT) can be stored at –70oC for several months. To process frozen lysates, thaw
samples for 15-20 minutes at 37oC in a water bath to dissolve salts.
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Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature.
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Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore NOT compatible with disinfecting reagents
containing bleach. Guanidine is an irritant. Take appropriate safety measures and wear gloves when handling.
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The bacterial culture should be harvested at 4oC. All subsequent steps of the RNeasy protocol should be performed at room
temperature. During the procedure, work quickly.
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After harvesting the cells, all centrifugation steps should be performed at 20-25oC in a standard microcentrifuge. Ensure that
the centrifuge does not cool below 20oC.
IV. Isolation of Total RNA from Bacteria
Prior to Harvesting Bacteria
1. Prepare 1 mg/ml lysozyme in TE Buffer (pH 8.0) for Gram-negative bacteria or 5 mg/mol lysozyme for
Gram-positive bacteria (150 µl per extraction).
Notes:
• A lysozyme stock solution (e.g. 20 mg/ml in water) can be made and stored in single-use
aliquots at –20oC.
• Add lysozyme stock solution to TE buffer just before use to make 1 mg/ml working solution.
2. Add 5 µl of β-ME to 495 µl of Buffer RLT for each RNA extraction
Notes:
• β-ME is toxic; dispense in a fume hood and wear appropriate protective clothing
• Buffer RLT is stable for 1 month after addition of β-ME
3. Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96100%), as indicated on the bottle, to obtain a working solution.
Harvesting Bacteria and Extracting RNA
1. Harvest bacteria by centrifuging at 5,000 x g for 5 minutes at 4oC.
Notes:
• The time and rcf required to pellet cells may differ among bacterial strains
• Do NOT use more than 1 x 109 bacteria
2. Decant supernatant, and carefully remove all remaining media by aspiration.
3. Loosen the bacterial pellet by flicking the bottom of the tube.
4. Resuspend the pellet in 150 µl of 1 mg/ml lysozyme (see above). Mix by vortexing, and incubate at
room temperature for 5 minutes. Vortex occasionally during incubation.
Notes:
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GENOMICS and SEQUENCING CENTER
• Depending on the bacterial strain used, the amount of lysozyme required and/or the
incubation time may vary.
• Complete digestion of the cell wall is essential for efficient lysis.
5. Add 400 µl of Buffer RLT to the sample. Mix thoroughly by vortexing vigorously.
Note: Ensure that β-ME is added to Buffer RLT before use (see above)
6. Add sample to a QIAshredder column placed in a 2 ml collection tube and centrifuge for 2 minutes at
full speed (14,000 rpm) in microfuge. Discard column after centrifugation.
7. Add 280 µl of ethanol (96-100%) to the lysate in the collection tube. Mix thoroughly by pipetting. Do
NOT vortex.
Note: A precipitate may form after the addition of ethanol, but this will not affect the RNeasy
procedure.
8. Apply the sample, including any precipitate that may have formed, to an RNeasy Mini column placed
in a 2 ml collection tube.
9. Close tube gently, and centrifuge for 15 seconds at 11,000 rpm. Discard the flow-through and place
column in a new 2 ml collection tube.
Note: If the volume exceeds 700 µl, load aliquots successively onto the RNeasy column, and
centrifuge as above. Discard flow-through after each centrifugation step.
10. Add 700 µl of Buffer RW1 into the RNeasy mini column.
11. Close the tube gently, and centrifuge for 15 seconds at 11,000 rpm to wash the column. Discard the
flow-through and place column in a new 2 ml collection tube.
12. Add 500 µl of Buffer RPE to the RNeasy mini column.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added prior to use (see
above).
13. Close the tube gently, and centrifuge for 15 seconds at 11,000 rpm to wash the column. Discard the
flow-through and place column in a new 2 ml collection tube.
14. Add another 500 µl of Buffer RPE to the RNeasy mini column.
15. Close the tube gently, and centrifuge for 2 minutes at 11,000 rpm to dry the RNeasy silica-gel
membrane. Discard the flow-through and place column in a 1.5 ml collection tube.
Notes:
• It is important to dry the RNeasy silica-gel membrane since residual ethanol may interfere
with downstream reactions.
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GENOMICS and SEQUENCING CENTER
• 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
16. Centrifuge at full speed (14,000 rpm) for 1 minute to eliminate any chance of possible Buffer RPE
carryover.
17. Place column in a new 1.5 ml collection tube.
18. To elute, add 50 µl of RNase-free water directly onto the RNeasy silica-gel membrane.
19. Close the tube gently, and centrifuge for 1 minute at 11,000 rpm.
20. If the expected RNA yield is >30 µg, repeat the elution in steps 18 and 19 with a second volume of
RNase-free water. Elute in the same collection tube.
Note: Alternatively, a higher total RNA concentration may be obtained if the 2nd elution is
performed using the first eluate (from step 19). The yield will be 15-30% less than the yield
obtained using a 2nd volume of RNase-free water, but the final concentration will be higher.
21. Store RNA at –70oC and avoid repeated freeze-thaw cycles.
Note: The purified RNA may contain a small amount of DNA contamination. DNA removal
may be necessary for certain RNA applications that are sensitive to very small amounts of DNA
(e.g. TaqMan RT-PCR analysis with a low abundance target). Qiagen offers an on-column
DNase digestion during RNA purification (see pgs. 99-100 of RNeasy Mini Handbook) or the
RNA can be treated with DNase after isolation.
[Created by Ryan Rhodes; July, 2004]
Rev.: 7/12/04
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