Transcript
Contents
Introduction
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
E.Z.N.A.® Total RNA Kit provides a rapid and easy method for the isolation of up to 100 : g of total RNA from cultured eukaryotic cells, tissues. The kit allows single or multiple, simultaneous processing of samples in less than 30 min. Normally, up to 1 x 107 eukaryotic cells or 30 mg tissue (amounts depend on the tissue used) can be used in a single experiment. There is no need for phenol/chloroform extractions, and time-consuming steps such as CsCl gradient ultracentrifugation, and precipitation with isopropanol is elim inated. W hile this kit may be used for isolation of RNA from whole blood, we recommend you use the E.Z.N.A.® Blood RNA Kit (product # R6614& R6814) as it is specifically designed for effective hemolysis and hem oglobin rem oval and gives higher RNA yields.
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Before Starting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Homogenization of Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.Liquid Nitrogen Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.Rotor-Stator Homogenizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.Beads Milling Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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RNA purified using the E.Z.N.A.® Total RNA method is ready for applications such as RT-PCR*, Northern blotting, poly A + RNA (mRNA) purification, nuclease protection, and in vitro translation.
Principle E.Z.N.A.® Total RNA Isolation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 A. Eukaryotic Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 B. Extraction of RNA from Fibrous Tissue . . . . . . . . . . . . . . . . . . . . . 8 Vacuum/Spin protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 RNA Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Quantization and Storage of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
The E.Z.N.A.® Total RNA Kits utilized the reversible binding properties of HiBind ® matrix, a new silica-based material, combined with the speed of mini-colum n spin technology to provide a fast and easy way for isolating total cellular RNA from cultured cells or animal tissues. A specifically formulated high salt buffer system allows more than 100 :g of RNA m olecules greater than 200 bases to bind to the m atrix. Cells or tissues are first lysed and homogenized under denaturing conditions that practically inactivate RNases. After adjust the binding condition by add ethanol, samples are then applied to the HiBind ® spin columns to which total RN A binds, while cellular debris and other contaminants are effectively washed away. High quality RNA is finally eluted in DEPC-treated sterile water.
RNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
New in this edition Troubleshooting Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
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Revised March 2005
On-column DNase I digestion protocol included. Optional vacuum-spin (for V-Spin column) protocol included.
Storage E.Z.N.A.® Total RNA Kits should be stored at room temperature. During shipm ent crystals may form in the TR K Lysis Buffer. Warm to 37o C to dissolve any precipitate. All E.Z.N.A.™ Total RNA Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22-25o C.
*The PCR proc ess is covered by U.S. Patents 4,683,195 and 4,683,202 (and international equivalents) owned by H offm ann- LaR oc he, Inc.
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This is normal and the bottle should be warmed to redissolve the
Kit Contents salt. Product Number
R6634-00 R6834-00
R6634-01 R6834-01
R6634-02 R6834-02
Purifications
5
50
200
HiBind™ Columns
5
50
200
10
100
400
T RK Lysis Bu ffer
5 ml
40 ml
1 50 m l
RN A W ash Bu ffer I
5 ml
40 ml
2 00 m l
RN A W ash Bu ffer II Con cen trate
5 ml
12 ml
50 ml
Disruption & Homogenization of Tissues
DEP C-ddH 2 O
1 ml
5 ml
20 ml
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A. Disrupt and Homogenize Tissue with Mortar and Pestle Wear gloves and take great care when working with liquid nitrogen. Excise tissue and promptly freeze in a small volume of liquid nitrogen. Grind tissue with a ceramic mortar and pestle under approximately 10 ml of liquid nitrogen and pour the suspension into a pre-cooled 15 ml polypropylene tube. Unless the tube is pre-cooled (in liquid nitrogen), the suspension will boil vigorously possibly causing loss of tissue. When the liquid nitrogen has completely evaporated, add TRK Lysis Buffer and continue with the procedure as outlined below. After interrupt tissue, lysate can be homogenized with Homogenizer Spin Column (Product # HCR 002). The lysate is loaded onto Homogenizer Spin Column in a 2 ml collection tube. Spin two minutes at a maximum speed in a microcentrifuge and the homogenized lysate is collected. Use the Omega Homogenizer Spin Column is a fast and efficient way to homogenize the lysate without cross contamination of samples. The alternated way to homogenize the lysate is to use the syringe and needle. High molecular-weight DNA can be sheared with by passing the lysate through a narrow needle (19-21 gauge) for 5-10 times.
2 m l Collection T ubes
Ins truc tion Man ual
Before Starting Wash Buffer II Concentrate must be diluted with absolute ethanol before use. IMPORTANT
Trial Sample (R6634-00/R6834-01)
Add 20 ml 100 % ethanol
R6634-01/R6834-01 R6634-02/R6834-02
Add 48 ml 100% ethanol Add 200 ml 100% ethanol
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2-mercaptoethanol (ß-mercaptoethanol) is key in denaturing RNases and must be added to an aliquot of TRK Lysis Buffer before use. Add 20 :l of 2-mercaptoethanol per 1 ml of TRK Lysis Buffer. This mixture can be stored for 1 week at room temperature.
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All centrifugation steps must be carried out at 22oC-25oC.
B. Disruption and Homogenization with Rotor-Stator Please take a few minutes to read this booklet thoroughly and become familiar with the protocol. Prepare all materials required before starting to minimize RNA degradation. !
Whenever working with RNA, always wear latex gloves to minimize RNase contamination. Use only clean RNase-free disposable plastic pipette tips when using the supplied reagents.
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During the procedure, work carefully but quickly.
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Under cool ambient conditions, crystals may form in TRK Lysis Buffer. 3
Rotor-Stator is the most preferred method for disruption and homogenizing tissue samples if required equipments are available. Rotor-stator homogenizers effectively homogenize and homogenize most tissues in the present of TRK Lysis Buffer . The process usually takes less than a minute depending on the type of tissue. Many Rotor-stator homogenizers operate with differently sized probes or generators that allow processing of small volumes in microfuge tubes. Such homogenizers are available from: ! ! !
Tekmar Inc., Cincinnati, OH (Tissuemizers®) BIOSPEC Products, Bartlesville, OK (Tissue-Tearor™) Craven Laboratories, Austin, TX.
C. Disruption and Homogenization with Beads Mills 4
Tissue sample can also be effectively disrupted and homogenized by rapid agitation in the presence of beads and lysis buffer. Tissue samples are disrupted and simultaneously homogenized with the sheared and crushed action of the beads as they collide with cells.
For tissue samples, homogenize using one of the methods discussed on page 4. For fatty tissues such as brain or adipose tissues, please use E.Z.N.A.® Total RNA Kit II (Product # R6934). For fibrous sample such as sample for muscle or heart, use protocol B in this user manual. Also OBI offers special designed kit for fibrous tissues (Product # R6688). Unless using liquid nitrogen, homogenize samples directly in TRK Lysis Buffer/2-mercaptoethanol and proceed to step 2.
E.Z.N.A.® Total RNA Isolation Protocol
A. Eukaryotic Cells and Tissues Materials supplied by user: ! 2-mercaptoethanol ! 70% ethanol in DEPC-treated sterile distilled water ! Sterile RNase-free pipette tips and microcentrifuge tubes ! Disposable latex gloves
2.
Add an equal volume (350 :l or 700 :l) 70% Ethanol to the lysate and mix thoroughly by vortexing.
3.
Apply sample onto HiBind® RNA spin column. The maximum capacity of the spin cartridge is 750 :l. (Larger volumes can be loaded successively.) A precipitate may form on addition of ethanol in step 2. Vortex and add the entire mixture to the column. With the spin column inside a 2ml collecting tube (supplied with kit), centrifuge at 10,000 x g for 15 seconds at room temperature. Discard flowthrough and collection tube.
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Place column in a clean 2ml collection tube, and add 300 :l RNA Wash Buffer I. Centrifuge and discard flow-through. Reuse the collection tube in step 6. If on-membrane DNase I digestion is desired, proceed step 5, otherwise go to step 6.
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DNase digestion (Optional) Since HiBind® RNA resin and spin-column technology actually removes most of DNA without the DNase treatment, it is not necessary to do DNase digestion for most downstream applications. However, certain sensitive RNA applications might require further DNA removal. Following steps provide on-membrane DNase I digestion:( see DNase I cat.# E1091for detail information)
Time Considerations: With E.Z.N.A.® Total RNA Kit,10 simultaneous samples can be prepared in approximately 25 min.
Procedure: 1.
Lyse cells or tissues with 350 :l of TRK Lysis Buffer. Remember to add 20 :l of 2-mercaptoethanol per 1 ml of TRK Lysis Buffer before use. 350 :l of TRK Lysis Buf fer is sufficient for# 5 x 106 cells or approximately 20 mg disrupted tissue (~3 mm cube). For difficult tissues, more than 5 x 106 cells, or greater than 20 mg tissue, use 700 :l of TRK Lysis Buffer. However, use no more than 30 mg tissue. For tissue culture cells grown in monolayer (fibroblasts, endothelial cells, etc.), lyse the cells directly in the culture vessel as follows. Aspirate culture medium completely and add TRK Lysis Buffer directly to the cells. Use 700 :l for T35 flasks or 10 cm dishes, and 350 :l for smaller vessels. Pipette buffer over entire surface of vessel to ensure complete lysis. Transfer lysate to a clean 1.5 ml microfuge tube and proceed to step 2 below. (This method is preferable to trypsinization followed by washing because it minimizes RNA degradation by nuclease contamination.)
a.
For cells grown in suspension cultures, pellet cells at no greater than 1,500 rpm (400 x g) for 5 min. Discard supernatant, add TRK Lysis Buffer, lyse by vortex or pipetting up and down, and transfer to a clean 1.5 ml microfuge tube. Proceed to step 2.
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prepare the DNase I
OBI DNase I Digestion Buffer
73.5 :l
RNase-free DNase I (20 Kunitz units/:l)
1.5 :l
Total volume
75 :l
Note: 1.
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For each HiBind ® RNA column, digestion reaction mix as follows:
DNase I is very sensitive for physical denaturaion, so do not vortex this DNase I mixture. M ix gently by inverting the tube. Prepare the fresh DNase I digestion mixture before RNA isolation.
2.
OBI DNase I digestion buffer is supplied with OBI RNase-free DNase set.
3.
Standard D Na se bu ff er s a re no t c om p at ib le wit h o n -m e m brane DNase digestion.
b. Pipet 75 :l of the DNase I digestion reaction mix directly onto the surface of HiBind® RNA resin in each column. Make sure to pipet the DNase I digestion reaction mixture directly onto the membrane. DNase I digestion will not be completed if some of the mix stick to the wall or the O-ring of the HiBind® RNA column. N
c. Incubate at room temperature(25-30 C) for 15 minutes 6.
Place column in a 2ml collection tube, and add 500 :l RNA W ash Buffer I. (If on-membrane DNase digestion was performed in the previous step, wait at least 5 minutes before proceeding). Centrifuge and discard flow-through.
7.
Place column in the same 2ml collection tube, and add 500 :l RNA Wash Buffer II diluted with ethanol. Centrifuge and discard flow-through. Reuse the collection tube in step 8.
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B. RNA Isolation from fibrous tissues (skeletal muscle, heart and aorta tissue, et al.) Materials supplied by user: ! 96-100% ethanol ! $-Mercaptoethanol ! RNase-free filter pipette tips ! Microcentrifuge capable of 12,000 xG ! Disposable latex gloves ! RNase-Free 1.5 ml tubes ! Water bath or heat block preset at 55°C ! Proteinase K Note: Equilibrate samples and TRK Lysis buffer to room temperature before start. All steps must be carried out at room temperature. Work quickly, but carefully.
1.
Excise tissue from animal or from storage.
Note: Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for instruction.
2.
Weight 10mg tissue and place it into a 1.5 ml tube for disruption and homogenization.
Wash column with a second 500 :l of Wash Buffer II as in step 7.
3.
Add 300 :l TRK lysis and disrupt tissue and homogenize lysate in TRK lysis buffer by using rotor-stator or mix beads mill system. Note: Incomplete homogenization will cause clogging of the spin column and lead to significantly lower yield. Generally, disruption and homogenization by using mortar and pestle or needle and syringe can generate lower yield. It is recommenced to use a rotor stator homogenizer or beads milling methods for animal tissues.
4.
Pipet 590 :l RNase-Feee water to each homogenate. Add 10 :l OB Protease and mix through by pipetting.
5.
Incubate at 55 °C for 10 minutes.
6.
Centrifuge at 10,000 x g for 5 minutes at room temperature. A small pellet of tissue debris will form and a thin layer or film can be seen on top of the supernatant.
7.
Transfer the supernatant into a RNase-Free 2 ml microtube. Avoid to transfer any of pellet. Hold the pipette tip under the thin
Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin cartridge for 1 min at full speed to completely dry the HiBind ® matrix. 9.
Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and elute the RNA with 50-100 :l of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 1 min at maximum speed. A second elution may be necessary if the expected yield of RNA >50 :g. Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 70oC before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.
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Pre-heating the water to 70oC before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.
layer or film on top of the supernatant, if present. This layer will usually adhere to outside the tip and should not be transferred. 8.
Add 450:l of absolute ethanol (96-100%) to the cleared lysate, mix throughly by pipetting.
9.
Place HiBind® RNA column into a 2ml collection tube (supplies) and carefully apply 700:l samples from step 8 (including any precipitate) to the HiBind® RNA column.
Vacuum/Spin Protocol for Isolation of RNA From Animal Cells (V-Spin column only) Use 350:l TRK Lysis Buffer, carry out lysis, homogenization, and loading onto HiBind ® RNA column as indicated previous protocols. Instead of continuing with centrifugation, follow steps blow. Do not use more than 106 cells or 10mg tissue for vacuum protocol.
10.
Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through and reuse the collection tube.
11.
Load the remaining of sample from step 8 and centrifuge at 10,000 x g for 30 seconds. Discard the flow-through and collection tube.
12.
DNase I Digestion (Optional): this is point to start Dnase digestion. (See detail protocol in page 6)
1.
Prepare the vacuum manifold according to manufacturer’ s instruction and connect the HiBind® RNA V-Spin column to the manifold.
13.
Place column in a clean 2ml collection tube, and add 500 :l RNA Wash Buffer I. (If on-membrane DNase digestion was performed in the previous step, wait at least 5 minutes before proceeding). Centrifuge and discard flow-through.
2.
Load the homogenized sample onto HiBind® RNA V-spin column.
3.
Switch on vacuum source to draw the sample through the column and turn off the vacuum.
4.
(Optional): Perform on-membrane DNase I digestion steps if sensitive
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Place column in the same 2ml collection tube, and add 500 :l RNA Wash Buffer II diluted with ethanol. Centrifuge and discard flow-through. Reuse the collection tube in step 7. Note: W ash Bu ffer II Con cen trate mu st b e diluted w ith abs olute ethanol bef ore us e. Ref er to label on bottle for instructions
15.
16.
Wash column with a second 500 :l of Wash Buffer II as in step 5. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin cartridge for 1 min at full speed to completely dry the HiBind ® matrix. Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and elute the RNA with 50-100 :l of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 1 min at maximum speed. A second elution may be necessary if the expected yield of RNA >50 :g. Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution.
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Note: Please read through previous section of this book before using this protocol.
downstream application is desired. (See previous section for details)
5.
Wash the column by adding 500 :l RNA wash buffer I, draw the wash buffer through the column by turn on the vacuum source.
6.
Wash the column by adding 700 :l RNA wash buffer II, draw the wash buffer through the column by turn on the vacuum source.
7.
Assemble the column into a 2 ml collection tube and transfer the column to a micro centrifuge. Add 200 :l of RNA wash buffer II into the column. Spin 2 minute to dry the column. Try to Avoid the carry over the RNA Wash Buffer II when remove the spin column from the collection tube.
8.
Place the column in a clean 1.5 ml microcentrifuge tube and add 50-100:l RNase-free water. Stand for 1-2 minute and centrifuge 1 minute to elute RNA.
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Trouble Shooting
RNA Contamination ®
Generally HiBind RNA spin column technology will efficiently removes m ost of the DNA without DNase treatment. However, no RNA extraction procedure can completely remove genomic DNA. For sensitive work (such as RT-PCR or differential display) we suggest that you perform on-column DNase I digestion (OBI cat# E1091) or treat the eluted RNA with RNase-free DNase. Also for RT-PCR, use intron-spanning prime rs that allow easy identification of DNA-contamination. A control PCR reaction containing the RNA as template will also allow detection of DNA contamination. For designing intron-spanning primers, call our technical staff at 800-832-8896 for assistance. W e can help design primers suited to your needs.
L ittle or no R N A eluted
RN A rem ains on t he c olu m n
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C log ged c olu m n
Quantization and Storage of RNA
C olum n is overloaded or clogg ed
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Reduce quantity of starting material.
Inc omp lete hom ogenization
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Com pletely homogenize sam ple. Inc rease c entrifu gation time. Reduce amount of starting material
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To determine the concentration and purity of RNA, m easure absorbance at 260 nm and 280 nm in a spectrophotometer. 1 O.D. unit measured at 260 nm corresponds to 40 :g of RNA per ml. DEPC-water is slightly acidic and can dramatically lower absorbance values. W e suggest that you dilute the sample in a buffered solution (TE) for spectro pho to metric analysis. The ratio of A260 /A 280 of pure nucleic acids is 2.0, while for pure protein it is approximately 0.6. A ratio of 1.8-2.0 corresponds to 90%-100% pure nucleic acid. (Phenol has an absorbance m aximum at 275 nm and can interfere with absorbance readings of DNA or RNA. However, the E.Z.N.A.® Total RN A Kit elim inates the use of phenol and avoids this problem.) Store RNA samples at -70o C in water. Under such conditions RNA prepared with the E.Z.N.A.® system is stable for m ore than a year.
! D eg rad ed R N A
Source
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! RNase con tamin ation
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RNA Quality It is highly recom mended that RNA quality be determined prior to all analyses. The quality of R NA can best be assessed by denaturing agarose gel electrophoresis and ethidium bromide staining. Two sharp bands should appear on the gel. These are the 28S and 18S (23S and 16S for bacteria) ribosom al RNA bands. If these band smear towards lower molecular weight RN As, then the RNA has undergone major degradation during preparation, handling, or storage. Although RNA molecules less than 200 bases in length do not efficiently bind the HiBind matrix, a third RNA band, the tRNA band, may be visible when a large number of cells are used.
Pr oblem in down stream applications
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Salt c arry-over dur ing elution
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! DNA con tamin ation
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Repeat elution. Pre- heat D EP C- water to 70 o C prior to elution. Inc ub ate colum n f or 10 min with w ater prior to centrifugation.
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Freeze s tarting m aterial quickly in liquid nitrogen. Do not store tissu e culture cells p rior to extraction unles s th ey are lysed firs t. Follow protocol closely, and wor k quic kly. Ensure not to introduce RN ase during the proc edure. Check buffers for RNase contamination. En su re W ash Bu ffer II Con cen trate has been diluted w ith 4 volum es of 100 % ethanol as indic ated on bottle. 1 X W ash Buffer II must be stored and u sed at room temp erature. Repeat wash with W ash Bu ffer II. Dig est w ith R Nas e-free DN ase and inactivate at 75 o C for 5 min.
Low A bs ratios
R N A d iluted in acidic buf fer or water
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D EP C -treated water is acid ic an d c an d ram atically lower Abs260 values. Use T E b uf fer to d ilu te R N A prior to sp ectr oph otom etric analysis.
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