GE Healthcare

illustra RNAspin Mini RNA Isolation Kit Product booklet Codes: 25-0500-70 (20 preps) 25-0500-71 (50 preps) 25-0500-72 (250 preps)

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Page finder 1. Legal

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2. Handling 2.1. Safety warnings and precations 2.2. Storage conditions 2.3. Expiry

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3. Components 3.1. Kit contents 3.2. Reagents to be supplied by user

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4. Description 4.1. The basic principle 4.2. Kit specifications

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5. Preparation of working solutions 5.1. RNase-free DNase I 5.2. Buffer RA3

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6. Handling, preparation, and storage of starting materials

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7. Protocols 7.1. Total RNA purification from cultured cells and tissue with RNAspin Mini Kit 7.2. Support protocol RNAspin Mini: Total RNA preparation from non-blood biological fluids (e.g., serum, culture medium) 7.3. Support protocol RNAspin Mini: Total RNA preparation from up to 109 bacterial cells 7.4. Support protocol RNAspin Mini: Total RNA preparation from up to 108 yeast cells 7.5. Support protocol—RNAspin Mini and RNAspin Midi: Clean-up of RNA from reaction mixtures

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8. Appendix 8.1. Troubleshooting

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1. Legal Product use restriction The RNAspin Mini Kit components have been designed, developed, and sold for research purposes only. They are suitable for in vitro uses only. No claim or representation is intended for its use to identify any specific organism or for clinical use (diagnostic, prognostic, therapeutic, or blood banking). It is the responsibility of the user to verify the use of the RNAspin Mini Kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism. GE and GE monogram are trademarks of General Electric Company. © 2006 General Electric Company – All rights reserved. GE Healthcare reserves the right, subject to any regulatory approval, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions http://www.gehealthcare.com GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK

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2. Handling or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice.

2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Buffers RA1, RA2 and MDB contain guanidine thiocyanate. Wear gloves and safety glasses.

2.2. Storage Store lyophilized RNase-free DNase I at +4ºC on arrival (stable up to 1 year). All other kit components should be stored at room temperature (20–25°C) and they are stable for up to one year. Storage at lower temperatures may cause precipitation of salts.

All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin

2.3. Expiry For expiry date please refer to outer packaging label.

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3. Components 3.1. Kit contents *Reagents highlighted with asterisk require prior preparation. Table 3.1. RNAspin Mini kit contents Pack Size 20 preps Cat. No. 25-0500-70 Buffer RA1 10 ml Buffer RA2 15 ml Buffer RA3 (concentrate)* 5 ml Buffer MDB (Membrane Desalting Buffer) 10 ml DNase reaction buffer 3 ml DNase I, RNase-free (lyophilized)* 1 vial H2O (RNase-free) 5 ml RNAspin Mini Filter units (violet) 20 RNAspin Mini columns (light blue-plus collection tube) 20 RNAspin Mini collection tubes 60 1.5 ml microcentrigue tubes 20 Protocol 1

50 preps 250 preps 25-0500-71 25-0500-72 25 ml 125 ml 15 ml 80 ml 12.5 ml 3 x 25 ml 25 ml 7 ml

125 ml 35 ml

1 vial

5 vial

15 ml

65 ml

50

250

50 150 50 1

250 750 250 1

3.2. Reagents to be supplied by user 70% and 95–100% ethanol β-mercaptoethanol

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4. Description 4.1 The basic principle

Fig 4.1. Overview of the RNAspin Mini procedure Figure 4.1. shows an overview of an RNA isolation procedure using the RNAspin Mini Kit. One of the most important aspects in the isolation process is to prevent the degradation of the RNA during 6

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the isolation procedure. With the RNAspin Mini method, cells are lysed by incubation in a solution containing large amounts of chaotropic ions. This lysis buffer immediately inactivates RNases, which are present in virtually all biological materials, and creates appropriate binding conditions that favor adsorption of RNA to the silica membrane. Contaminating DNA, which is also bound to the silica membrane, is removed by the direct application of a DNase I solution to the silica membrane (RNase-free DNase I is supplied with the kit). Simple washing steps with two different buffers remove salts, metabolites and macromolecular cellular components. Finally, pure RNA is eluted under low ionic strength conditions with RNasefree water (supplied). RNA isolation using the RNAspin Mini Kit can be performed at room temperature. However, the eluate should be treated with care because RNA is very sensitive to trace contaminations of RNases, often found on general labware, fingers and dust. To preserve stability, keep the isolated total RNA frozen at -20°C for short-term or -80°C for long-term storage.

4.2 Kit specifications The RNAspin Mini Kit is recommended for the isolation of total RNA from cultured cells and tissue. The kit can be used to isolate RNA from different amounts of sample material according to Table 4.1.

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Table 4.1. Examples of Input Sample Material Sample

amount

Cultured animal cells (e.g., HeLa cells)

up to 5 x 106

Animal tissue

up to 30 mg

Bacteria

up to 1 x 109

Yeast

up to 5 x 107

RNAspin Mini Kits allow for the isolation of pure RNA with an A260/A280 ratio generally exceeding 1.9 (measured in 0.1 M Tris-HCl buffer (pH 7.6)). Even biological samples that are sometimes difficult to process, will yield high quality RNA. These include mouse tissue (liver, brain), different tumor cell lines, Streptococci, and Actinobacillus pleuropneumoniae. Note that this kit is not suitable for isolation of RNA from blood. The isolated RNA is ready to use for downstream applications like quantitative Reverse Transcriptase-PCR (QRT-PCR), Primer Extension, RNase Protection Assays, cDNA Synthesis and Microarray Analysis.

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A

B

28S 18S

Fig. 4.2 The RNAspin Mini kits produce high quality RNA: rRNA bands are sharp, with the 28S band being about twice as intense as the 18S band, and with good RNA Integrity Number (RIN) values. (A) Total RNA from 106 HeLa cells was isolated with RNAspin Mini and separated by gel electrophoresis on a 1.2% formaldehyde agarose gel; (B) Total RNA from rat liver was isolated with RNAspin Mini and evaluated using the Agilent 2100 bioanalyzer.

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Table 4.2. RNAspin Mini Technical Specifications at a Glance Animal tissue

Cell culture

Sample size

up to 30 mg tissue

up to 5 x 106 cells

Typical yield

up to 70 μg

up to 70 μg

Elution volume

40–120 μl

Effective binding capacity RNA integrity

100 μg sharp rRNA bands with no substantial degradative bands visible 28S:18S = ~2:1 RNA Integrity Number (RIN) values ≥7

RNA purity

A260/A280 = 1.8–2.2

Time/Prep

1 x 106) or tissue (>10 mg), first homogenize using a 0.9 mm needle (20 gauge), followed by filtration through RNAspin Mini Filter units. 2. Cell lysis Add 350 μl buffer RA1 and 3.5 μl β-mercaptoethanol to the cell pellet or to ground tissue and vortex vigorously 3. Filtration of the lysate Reduce viscosity and clear the lysate by filtration through RNAspin Mini Filter units: Place RNAspin Mini Filter units (violet ring) in a collection tube, apply the mixture, and centrifuge for 1 min at 11 000 x g.

+ 350 μl RA1 + 3.5 μl β-ME

1 min 11 000 x g

Alternatively, the lysate may be passed ≥ 5 times through a 0.9 mm needle (20 gauge) fitted to a syringe.

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Discard the RNAspin Mini Filter. Transfer filtrate, taking care to avoid aspirating any formed pellet, to a new 1.5 ml microcentrifuge tube (not included). 4. Adjust RNA binding conditions Add 350 μl ethanol (70%) to the homogenized lysate and mix by vortexing, 2 x 5 sec. After the addition of ethanol, a stringy precipitate may become visible. This will not affect RNA isolation. Be sure to load all of the precipitate onto the column as described in step 5. 5. Bind RNA For each preparation, use one RNAspin Mini column (light blue ring) placed in a 2 ml microcentrifuge tube. Pipet lysate up-and-down 2–3 times, and then load the lysate onto the column. Centrifuge for 30 s at 8000 x g. Place the column in a new collection tube.

+ 350 μl 70% EtOH

mix

load lysate

30 sec 8000 x g

Maximal loading capacity of RNAspin Mini column is 750 μl. Repeat the procedure if larger volumes are to be processed. 6. Desalt silica membrane Add 350μl MDB (Membrane Desalting Buffer) and centrifuge at 11 000 x g for 1 min to dry the membrane. Discard the flow-through and return the column to the collection tube.

+350 μl MDB

1 min 11 000 x g

Salt removal will make the subsequent DNase I digest much more effective. If the column outlet has come into contact with the flow-through 15

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for any reason, discard the flow-through and centrifuge again for 30 sec at 11 000 x g. 7. Digest DNA Prepare DNase reaction mixture in a sterile microcentrifuge tube: for each isolation, add 10μl reconstituted DNase I (from section 5) to 90μl DNase reaction buffer. Mix by flicking the tube.

+ 95 μl DNase reaction mixture RT 15 min

Apply 95 μl of DNase reaction mixture directly onto the center of the silica membrane of the column. Incubate at room temperature for 15 min. 8. Wash and Dry silica membrane Make sure buffer RA3 is equilibrated to room temperature. 1st wash Add 200μl buffer RA2 to the RNAspin Mini column. Centrifuge for 1 min at 11 000 x g. Place the column into a new collection tube.

+ 200 μl RA2

1 min 11 000 x g

Buffer RA2 will inactivate DNase I.

2nd wash Add 600 μl buffer RA3 to the RNAspin Mini column. Centrifuge for 1 min at 11 000 x g. Discard flow-through and place the column back into the collection tube.

+600 μl RA3

1 min 11 000 x g

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3rd wash Add 250 μl buffer RA3 to the RNAspin Mini column. Centrifuge for 2 min at 11 000 x g to dry the membrane completely. Place the column into a nuclease-free 1.5 ml microcentrifuge tube (supplied).

+250 μl RA3

2 min 11 000 x g

If for any reason the liquid level in the collection tube has reached the RNAspin Mini column after centrifugation, discard flow-through and centrifuge again. Check that no residual flow-through remains in the column outlet. If any remains, re-centrifuge. 9. Elute highly pure RNA Elute the RNA in 100 μl H2O (RNase-free; supplied) and centrifuge at 11 000 x g for 1 min. It is possible to adapt the elution protocol for the subsequent application of interest.

+ 100 μl H2O (RNase-free)

1 min 11 000 x g

High yield: Perform two elution steps with the volume indicated in the individual protocol. About 90–100% of bound nucleic acid will be eluted. High concentration: Elute with a 40 μl elution volume. High yield and high concentration: Elute with the standard elution volume and apply the eluate a second time onto the column for re-elution. Eluted RNA should be immediately placed on ice to prevent potential degradation. Keep at -20°C and -80°C for short-and long-term storage, respectively. 17

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7.2. Support protocol RNAspin Mini: Total RNA preparation from non-blood biological fluids (e.g., serum, culture medium) No need for homogenization or filtration of the lysate. 1. Cell lysis Add 350 μl buffer RA1 to 100 μl of sample and vortex vigorously . 2. Adjust RNA binding conditions Add 350 μl of ethanol (70%) to the lysate and mix by vortexing, 2 x 5 sec Proceed with step 5 of the RNAspin Mini standard protocol (section 7.1).

7.3. Support protocol RNAspin Mini: Total RNA preparation from up to 109 bacterial cells 1. Homogenization of sample Resuspend the bacterial cell pellet (Gram-negative strains) in 100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8) containing 0.2 mg/ml lysozyme by vigorous vortexing. Incubate at 37°C for 10 min. For preparation of RNA from Gram-positive bacteria, resuspend cells in 100 μl TE containing 2 mg/ml lysozyme. It may be necessary to optimize incubation time and lysozyme concentration, according to the bacterial strain. 2. Cell lysis Add 350 μl buffer RA1 and 3.5 μl β-mercaptoethanol to the suspension and vortex vigorously. 3. Filtration of lysate Reduce viscosity and turbidity of the solution by filtration through RNAspin Mini Filter units. Place the RNAspin Mini Filter units in

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collection tubes, apply mixture, and centrifuge for 1 min at 11 000 x g. Alternatively, the lysate may be passed ≥ 5 times through a 0.9 mm needle (20 gauge) fitted to a syringe. 4. Adjust RNA binding conditions Add 350 μl of ethanol (70%) and proceed with step 5 of the RNAspin Mini standard protocol (section 7.1). Because of the higher relative concentration of genome equivalents in a nucleic acid preparation of bacteria compared with eukaryotic material, it may be necessary to use a lower amount of cells for the preparation.

7.4. Support protocol RNAspin Mini: Total RNA preparation from up to 5 x 108 yeast cells 1. Homogenization of sample Harvest 2–5 ml of YPD culture (centrifugation at 5000 x g, 10 min). Decant culture media. Resuspend pellet in sorbitol/lyticase buffer (50–100 U lyticase or zymolase in 1 M sorbitol/100 mM EDTA) and incubate at 30°C for 30 min. Pellet the resulting spheroplasts by centrifugation (1000 x g, 10 min). It may be necessary to optimize incubation time and lyticase/ zymolase concentration, according to the yeast strain. 2. Cell lysis Add 350 μl buffer RA1 and 3.5 μl β-mercaptoethanol to the suspension and vortex vigorously. 3. Filtration of lysate Reduce viscosity and turbidity of the solution by filtration through RNAspin Mini Filter units. Place RNAspin Mini Filter units in collection tubes, apply mixture, and centrifuge for 1 min at 11 000 x g.

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Alternatively, the lysate may be passed ≥5 times through a 0.9 mm needle (20 gauge) fitted to a syringe. Proceed with step 4 of the RNAspin Mini standard protocol (section 7.1) Due to the higher relative concentration of genome equivalents in a nucleic acid preparation of yeasts compared with cultured cells or tissue material, it may be necessary to use a lower amount of cells for the preparation.

7.5. Support protocol - RNAspin Mini and RNAspin Midi: Clean-up of RNA from reaction mixtures 1. Clean-up Add 3.5 volumes of buffer RA1 per 1 volume of sample, and vortex to mix. Maximal loading capacity of RNAspin Mini columns is 750 μl. Repeat the procedure if larger volumes are to be processed. In order to load all of the sample in subsequent step 5 in one step: - do not use more than 90 μl of sample for the RNAspin Mini Kit, - do not use more than 480 μl of sample for the RNAspin Midi Kit. 2. Adjust RNA binding conditions Add ethanol (95–100%) at 3.5 times the volume of sample only from step 1 to the lysate, and mix by vortexing. Proceed with step 5 of section 7.1 of either the RNAspin Mini or the RNAspin Midi standard protocols.

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8. Appendix 8.1. Troubleshooting guide Problem: RNA is degraded/no RNA obtained Possible cause Suggestions RNase contamination

• Create an RNase-free working environment. Wear gloves during all steps of the procedure, and change gloves frequently. Use of sterile, disposable polypropylene tubes is recommended. Keep tubes closed whenever possible during the preparation. Glassware should be ovenbaked for at least 2 hours at 250°C before use.

Problem: Poor RNA quality or yield Possible cause Suggestions Reagents not prepared, stored or applied properly

Reagents not properly stored. Add the indicated volume of nuclease-free water to DNase I vial and 96% ethanol to buffer concentrate RA3 and mix. Reconstitute and store lyophilized DNase I according to instructions given in section 5. • Sample and reagents have not been mixed completely. Always vortex vigorously after each reagent has been added. • Ethanol was not added after lysis. Binding of RNA to the silica membrane is only effective in the presence of ethanol.

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Problem: Poor RNA quality or yield Possible cause Suggestions Reagents not • Reconstitute and store lyophilized prepared, stored or DNase I according to instructions given applied properly, in section 5. continued • Store other kit components at room temperature. Storage at low temperatures may cause salt precipitation. • Keep bottles tightly closed in order to prevent evaporation or contamination. Suboptimal elution

• Be sure that all of the water gets into contact with the silica membrane. No water drops should stick to the walls of the columns. The membrane has to be wetted completely. Ionic strength and pH influence A260 absorption as well as ratio A260/A280; thus, for absorption measurement, use 0.1 M Tris-HCl pH 7.6 as diluent.

Sample material • Sample material not stored properly. Whenever possible, use fresh material. If this is not possible, flash freeze the samples in liquid N2 or treat with a stabilizing agent. Samples should always be kept at -80°C. Never allow tissues to thaw before addition of buffer RA1. Perform disruption of samples in liquid N2, if possible.

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Problem: Poor RNA quality or yield. continued Possible cause Suggestions Sample material, • Insufficient disruption and/or continued homogenization of starting material. Ensure thorough sample disruption and use RNAspin Mini Filter for easy clean-up of disrupted starting material. • Too much starting material may lead to RNA Binding column clogging or reduced RNA quality or yield. For clogging issues, see below. RNA quality and yield problems relating to too much sample material may be addressed by decreasing the amount of starting material and/or increasing the volumes of wash buffers RA2 and RA3. Problem: Clogged RNAspin Binding Column Possible cause Suggestions Sample material • Use the RNAspin Mini Filter to reduce the risk of clogging the Binding column. • To prevent clogging due to too much starting material, reduce the sample amount, increase the time for the centrifugation steps, and/or increase the volume of buffer RA1. If clogging still occurs during the run, take the remaining lysate off the RNAspin RNA Binding column, discard it, and proceed with the desalting step (with buffer MDB).

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Problem: Contamination of RNA with genomic DNA Possible cause Suggestions DNase I not active • Reconstitute and store lyophilized DNase I according to instructions given in section 5. DNase solution not properly applied

• Pipette DNase I solution directly onto the center of the silica membrane.

Too much cell material used

• Reduce quantity of cells or tissue used

DNA detection system too sensitive

• The amount of DNA contamination is significantly reduced during the oncolumn DNase I digestion. Residual DNA may still be present; therefore in very sensitive applications, it might be possible to detect DNA. As part of the product QC process, the RNAspin Mini system is assessed for DNA contamination by the following procedure: 1 x 106 HeLa cells are subjected to RNA isolation according to the protocol. RNA eluate is used as template for PCR detection of a 1 kb fragment in a 30cycle reaction. Generally, no PCR fragment is obtained if DNase I is applied, however, a strong PCR fragment is obtained if the DNase is omitted.

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Problem: Contamination of RNA with genomic DNA, continued Possible cause Suggestions DNA detection system The potential for DNA contamination too sensitive detection by PCR increases with: continued • the number of DNA copies per preparation: single copy target < plastidial/mitochondrial target < plasmid transfected into cells

• decreasing PCR amplicon size: use larger PCR targets (e.g. >500 bp) or intron-spanning primers if possible. Problem: Suboptimal performance of RNA in downstream experiments Possible cause Suggestions Carry-over of ethanol • Do not let the flow-through touch or salt the column outlet after the second RA3 wash. Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic buffer RA3 completely. • Check if buffer RA3 has been equilibrated to room temperature before use. Washing at lower temperatures lowers efficiency of salt removal by RA3. Store isolated RNA properly

• Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases will degrade the isolated RNA. For shortterm storage freeze at -20°C, for longterm storage, freeze at -80°C. 25

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GE Healthcare regional office contact numbers:

Germany Tel: (089) 96281 660 Fax: (089) 96281 620

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UK

Fax: 02 416 82 06

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http://www.gehealthcare.com/lifesciences GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK

25-0500-70PL Rev A 2006

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The next two pages are a protocol card. Please add to the back page as a tear off addition.

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illustra RNAspin Mini RNA Isolation Kit Product protocol card

25-0500-70

1. Homogenization of sample