RNA Isolation Kit Instruction Manual

RNA Isolation Kit Instruction Manual
RNA Isolation Kit
Instruction Manual
Catalog #200345
Revision B
Research Use Only. Not for Use in Diagnostic Procedures.
200345-12
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RNA Isolation Kit
CONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions .............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 1
Introduction......................................................................................................................................... 2
Protocol Guidelines ............................................................................................................................. 2
RNA Isolation Protocols ..................................................................................................................... 3
Homogenizing Animal Tissue ............................................................................................... 3
Preparing Tissue Culture Cells Grown in Suspension........................................................... 3
Preparing Tissue Culture Cells Grown in Monolayer ........................................................... 4
Isolating RNA........................................................................................................................ 5
Alternative Time-Saving Protocol for Isolating RNA........................................................... 6
Troubleshooting .................................................................................................................................. 8
Appendix I: Spectrophotometric Quantitation of RNA ................................................................ 10
Appendix II: Formaldehyde Gel Protocol ...................................................................................... 11
Preprotocol Considerations ................................................................................................. 11
Protocol ............................................................................................................................... 11
Expected Results ................................................................................................................. 12
Appendix III: Northern Transfer Protocol..................................................................................... 13
Preprotocol Considerations ................................................................................................. 13
Protocol ............................................................................................................................... 13
Appendix IV: Hybridization Protocol ............................................................................................. 16
Preprotocol Considerations ................................................................................................. 16
Protocol ............................................................................................................................... 16
Appendix V: Avoiding DNA Contamination .................................................................................. 18
Preparation of Media and Reagents ................................................................................................ 19
References .......................................................................................................................................... 20
Endnotes............................................................................................................................................. 20
MSDS Information............................................................................................................................ 20
Quick-Reference Protocol ................................................................................................................ 21
RNA Isolation Kit
MATERIALS PROVIDED
Materials Provideda
Concentration
Denaturing solution
—
100 ml
β-Mercaptoethanol
14.33 M
750 μl
2M
7.5 ml
Sodium acetate (pH 4.0)
Phenol (pH 5.3–5.7, equilibrated with 0.1 M succinic acid)
a
b
c
Quantity
—
75 ml
Chloroform–isoamyl alcohol
—
15 ml
Isopropanol
—
100 ml
b,c
The RNA Isolation Kit provides enough reagents to isolate total RNA from 7 g of tissue or from 7 × 108 tissue
culture cells.
The phenol provided with this kit is shipped at room temperature.
Do not use the phenol provided with this kit for DNA isolation.
STORAGE CONDITIONS
β-Mercaptoethanol: 4°C
Phenol (Equilibrated with Succinic Acid): 4°C
All Other Components: Room Temperature
Warning
The denaturing solution contains the irritant guanidine isothiocyanate.
ADDITIONAL MATERIALS REQUIRED
Tissue grinder with a Teflon® pestle and a glass receptacle or a rotating blade homogenizer such as a
Polytron® homogenizer
Polypropylene tubes (50 ml)
Ethanol [75% (v/v)] in diethylpyrocarbonate(DEPC)-treated water
DEPC-treated water
Revision B
RNA Isolation Kit
© Agilent Technologies, Inc. 2010.
1
INTRODUCTION
The RNA Isolation Kit minimizes the major difficulty in isolating RNA: the
degradation of RNA by ribonucleases (RNases). The kit uses guanidine
isothiocyanate1 (GITC), one of the strongest protein denaturants, to
inactivate RNases and protect the RNA from degradation. High yields of
high-purity, intact, total RNA are recovered from tissues rich in RNases,
from a small amount of tissue, or from a small number of tissue culture
cells. The quality of the isolated RNA is sufficient to construct a cDNA
library and to isolate a desired clone. The kit uses a single-step, GITC,
phenol–chloroform extraction,2 which allows isolation of RNA in only
4 hours with the standard protocol or in only 60–90 minutes with the
alternative time-saving protocol. The method limits handling of the sample
and materials, which reduces contamination of samples by RNases and
consequent loss and degradation of RNA. Because of the simplicity of the
technique and the elimination of ultracentrifugation steps, multiple samples
can be run simultaneously.
PROTOCOL GUIDELINES
♦ Wear gloves at all times during the extraction procedure and while
handling materials and equipment to prevent contamination by RNases.
♦ Exercise care to ensure that all equipment (e.g., the homogenizer,
centrifuge tubes, etc.) is as free as possible from contaminating RNases.
Treatment of all equipment with diethyl-pyrocarbonate (DEPC)3 and
autoclaving with baking is recommended for all equipment.
♦ Prepare all solutions with DEPC-treated water, using 0.1% (v/v) DEPC
in distilled water (dH2O). Allow the DEPC-treated water to incubate
overnight at room temperature and then autoclave the DEPC-treated
water prior to use. If a solution contains Tris base, prepare the solution
with DEPC-treated water and autoclave the solution before adding Tris
base. Autoclave the solution once again after the addition of Tris base to
the solution (see Preparation of Media and Reagents).
2
RNA Isolation Kit
RNA ISOLATION PROTOCOLS
Homogenizing Animal Tissue
This protocol is for isolating total RNA from 1 g of animal tissue.
Note
To reduce RNA degradation, extract RNA from the tissue
immediately after dissection from the animal or flash-freeze the
tissue sample in liquid nitrogen immediately after dissection.
1. Prepare solution D by adding 100 μl of β-mercaptoethanol to 14 ml of
room temperature denaturing solution. Solution D may be stored at
room temperature for up to 1 month.
2. Quickly weigh 1 g of tissue and immediately store the remaining tissue
in liquid nitrogen. Immediately place the tissue sample into a tube
containing 10 ml of solution D.
3. Mince and homogenize the tissue with 10 ml of solution D in a glass–
Teflon homogenizer. Transfer the homogenate to a 50-ml
polypropylene tube. Alternatively, homogenize the tissue directly in a
50-ml polypropylene tube with a rotating blade homogenizer such as
the Polytron homogenizer or an equivalent instrument. Save the
remaining 4 ml of solution D on ice until needed in step 11 of Isolating
RNA.
4. Proceed with the protocol in Isolating RNA or with the Alternative
Time-Saving Protocol for Isolating RNA.
Preparing Tissue Culture Cells Grown in Suspension
This protocol is for isolating total RNA from 1 × 108 tissue culture cells
grown in suspension. Vary the volume of the reagents proportionately to the
number of cells in the sample.
1. Prepare solution D by adding 100 μl of β-mercaptoethanol to 14 ml of
denaturing solution. Solution D may be stored at room temperature for
up to 1 month.
2. Gently pellet the cells by centrifugation and discard the supernatant.
3. Add 10 ml of solution D to the cell suspension and mix thoroughly.
Save the remaining 4 ml of solution D on ice until needed once again in
step 11 of Isolating RNA.
4. Incubate the suspension for 1 minute at room temperature.
5. Transfer the cell suspension to a 50-ml polypropylene tube.
6. Proceed with the protocol in Isolating RNA or the Alternative TimeSaving Protocol for Isolating RNA.
RNA Isolation Kit
3
Preparing Tissue Culture Cells Grown in Monolayer
This protocol is for isolating total RNA from tissue culture cells grown in
monolayer and can be used for a maximum of ten 100-mm tissue culture
dishes.
4
1.
Prepare solution D by adding 100 μl of β-mercaptoethanol to 14 ml of
denaturing solution. Solution D may be stored at room temperature for
up to 1 month.
2.
Decant the tissue culture medium from the tissue culture plates.
3.
Add 10 ml of solution D to a tissue culture plate and swirl the plate
gently for 30 seconds. Store the remaining 4 ml of solution D on ice
until needed in step 11 of Isolating RNA. (For a single tissue culture
plate, use 2 ml of solution D and divide the volumes of the reagents
used in the remainder of the protocol by 5.)
4.
Using a pipet, transfer solution D from the tissue culture plate to a
second tissue culture plate and swirl the second plate gently for
30 seconds.
5.
Repeat step 4 for the remaining plates.
6.
Transfer the cell–solution D mixture in the final plate to a 50-ml
polypropylene tube.
7.
Proceed with the protocol in Isolating RNA or the Alternative TimeSaving Protocol for Isolating RNA.
RNA Isolation Kit
Isolating RNA
1. Add 1.0 ml of 2 M sodium acetate (pH 4.0) to the sample. Mix the
contents of the tube thoroughly by repeatedly inverting the tube.
2. Add 10.0 ml of phenol (pH 5.3-5.7) to the tube. Mix the contents
thoroughly by inversion.
Note
The provided phenol is equilibrated with succinic acid and is
contained in the bottom phase of the bottle (under the
aqueous phase). Take care to pipet the bottom phase
containing the phenol and to avoid the aqueous phase. If the
phenol and aqueous phases are not clear and distinct,
incubate the phenol at room temperature or heat the phenol
at 37°C until two distinct phases are present.
3. Add 2.0 ml of chloroform–isoamyl alcohol mixture to the sample. Cap
the tube tightly and shake the tube vigorously for 10 seconds.
4. Incubate the tube on ice for 15 minutes.
5. Transfer the sample to a 50-ml, thick-walled, round-bottom centrifuge
tube that has been prechilled on ice.
6. Spin the tube in a centrifuge at 10,000 × g for 20 minutes at 4°C.
Note
After centrifugation, two phases should be clearly visible. The
upper, aqueous phase contains the RNA. The lower, phenol
phase and interphase contain DNA and proteins.
7. Taking care to avoid the interphase and the lower phase, transfer the
upper, aqueous phase, which contains the RNA, to a fresh centrifuge
tube. Discard the lower phenol phase, which contains proteins and
DNA.
8. Add an equal volume of isopropanol to the tube that contains the
aqueous phase and mix the contents by inversion.
9. Incubate the tube for ≥1 hour at –20°C to precipitate the RNA.
10. Spin the tube in a centrifuge at 10,000 × g for 20 minutes at 4°C. After
centrifugation, the pellet at the bottom of the tube contains the RNA.
Remove and discard the supernatant.
11. Dissolve the pellet in 3.0 ml of solution D. Gently pipet the pellet, if
necessary.
12. Add 3.0 ml of isopropanol to the tube and mix the contents well.
13. Incubate the tube for 1 hour at –20°C.
RNA Isolation Kit
5
14. Spin the tube in a centrifuge at 10,000 × g for 10 minutes at 4°C.
Remove and discard the supernatant from the tube.
Note
Up to this point, the RNA has been protected from
ribonucleases by the presence of guanidine isothiocyanate.
The RNA is now no longer protected.
15. For applications in which the salt concentration is important, wash the
pellet with 75% (v/v) ethanol [DEPC-treated water (25%)]. Remove
and discard the supernatant.
16. Dry the pellet under vacuum for 2–5 minutes. Do not over dry the
sample because an over dry pellet will be difficult to resuspend.
17. Resuspend the RNA in 0.5–2 ml of DEPC-treated water. The A260/A280
ratio should be ≥1.8 (see Appendix I: Spectrophotometric Quantitation
of RNA).
Polyadenylated RNA [poly(A)+] can now be isolated from the total RNA for
use in subsequent cDNA synthesis and cloning, northern blot analysis, or
translation studies.
For long-term storage, resuspend the RNA in DEPC-treated water, add 2 M
sodium acetate to a final concentration of 0.25 M, and then add 2.5 volumes
of 100% (v/v) ethanol. Store the RNA at –80°C.
Note
When ready to use the RNA after long-term storage in ethanol,
repeat steps 14–17.
Alternative Time-Saving Protocol for Isolating RNA
This time-saving protocol is an alternative protocol to Isolating RNA and is
performed in 60–90 minutes.
1. Add 1.0 ml of 2 M sodium acetate (pH 4.0) to the sample. Mix the
contents of the tube thoroughly by repeatedly inverting the tube.
2. Add 10.0 ml of phenol (pH 5.3-5.7) to the tube. Mix the contents
thoroughly by inversion.
Note
The provided phenol is equilibrated with succinic acid and is
contained in the bottom phase of the bottle (under the
aqueous phase). Take care to pipet the bottom phase
containing the phenol and to avoid the aqueous phase. If the
phenol and aqueous phases are not clear and distinct,
incubate the phenol at room temperature or heat the phenol
at 37°C until two distinct phases are present.
3. Add 2.0 ml of chloroform–isoamyl alcohol mixture to the homogenate.
Cap the tube tightly and shake the tube vigorously for 10 seconds.
4. Transfer the homogenate to a 50-ml, thick-walled, round-bottom
centrifuge tube.
6
RNA Isolation Kit
5. Spin the tube in a centrifuge at 12,000 × g for 15 minutes at 5–10°C.
Note
After centrifugation, two phases should be clearly visible. The
upper aqueous phase contains the RNA. The lower phenol
phase and interphase contain DNA and proteins.
6. Taking care to avoid the interphase and the lower phase, transfer the
upper, aqueous phase, which contains the RNA, to a sterile, RNasefree, 50-ml, thick-walled, round-bottom centrifuge tube. Discard the
lower phenol phase, which contains proteins and DNA.
7. Add an equal volume of isopropanol to the tube that contains the
aqueous phase and mix the contents by inversion.
8. Spin the tube in a centrifuge at 10,000 × g for 30 minutes at 5–10°C.
9. Remove and discard the supernatant.
Note
Up to this point, the RNA has been protected from
ribonucleases by the presence of guanidine isothiocyanate.
The RNA is now no longer protected.
10. Wash the pellet with 75% (v/v) ethanol [DEPC-treated water (25%)].
11. Remove and discard the supernatant. Dry the pellet under vacuum for
2–5 minutes. Do not over dry the sample because an over dry pellet
will be difficult to resuspend.
12. Resuspend the RNA in 0.5–2 ml of DEPC-treated water. The A260/A280
ratio should be ≥1.8 (see Appendix I: Spectrophotometric Quantitation
of RNA).
RNA Isolation Kit
7
TROUBLESHOOTING
Observation
Suggestion(s)
Interphase layer is not well resolved
Ensure that β-mercaptoethanol was added to the denaturing solution
Ensure that chloroform–isoamyl alcohol solution was added to the sample
Chloroform may have evaporated from the chloroform-isoamyl alcohol mixture,
altering the ratio of chloroform to alcohol. When this occurs, phase separation is
reduced. Add a few drops of chloroform to the extraction mixture, mix briefly by
inversion, then recentrifuge the sample
Ensure that sodium acetate was added to the sample. If sodium acetate was
omitted from the extraction mixture, it may be added later to induce phase
separation. Add the 1.0 ml of sodium acetate to the extraction mixture, mix by
inversion, then recentrifuge the sample
Remove the aqueous layer immediately after centrifugation, as the phases become
less well resolved over time. Centrifuge the samples again for 5 minutes
Unable to resuspend the RNA pellet
after precipitation
Increase the temperature of the final resuspension to 65°C for 5 minutes and
increase the volume of the DEPC-treated water
Vortex the pellet gently
Add additional DEPC-treated water and allow the RNA pellet to dissolve for a
longer time
Degraded RNA
The sample may be contaminated with RNase. Repeat the protocol, focusing strict
attention on avoiding the introduction of RNases from hands and/or other
contaminated surfaces, especially after step 14 of the protocol. At this stage the
RNA pellet is particularly vulnerable to contaminating RNases
Use DEPC-treated or radiation-sterilized plasticware.
Introduce the tissue or tissue culture cells into solution D at the earliest possible
moment to ensure inactivation of contaminating RNases
If isolating the RNA from tissue, flash-freeze the tissue immediately after dissection
from the animal. Some tissues, such as the spleen and pancreas, are very difficult
to process without RNase degradation and must be flash-frozen immediately after
isolation from the animal
If isolating the RNA from cultured cells, remove cultured cells from the incubator at
the last possible moment, process the cells rapidly once the medium is removed
The final optical density has an
absorbance ratio (A260/A280) <1.8
The starting ratio of tissue or tissue culture cells to solution D is >100 mg/ml.
Adjust the starting ratio of tissue or tissue culture cells to ~100 mg/ml
During the phenol extraction, a portion of the middle and lower phases is removed
along with the upper, aqueous phase that contains the RNA. Carefully avoid the
middle and lower phases of the extraction while transferring the upper aqueous
phase to a sterile, 50-ml polypropylene tube
Avoid using DEPC-treated water in the spectrophotometric analysis as it skews the
260:280 absorbance ratio
(Table continues on the next page)
8
RNA Isolation Kit
(Table continued from the previous page)
Note that RNA yields vary depending on the tissues or cell lines employed
The yield of RNA is lower than
expected
The membranes of the tissue or tissue culture cells are not disrupted completely.
Homogenize the tissue sample completely in solution D or disrupt the tissue culture
cells by swirling or mixing thoroughly to release the nucleic acids
The aqueous phase is not removed completely following extraction
Some of the RNA pellet is decanted following the ethanol wash. Because residual
ethanol can be removed from the RNA pellet via air-drying or under vacuum,
carefully decant the ethanol wash to avoid discarding any of the RNA pellet
The spectrophotometric measurement is inaccurate. Use ≥1μg of RNA in the
spectrophotometric measurement
The RNA pellet is resuspended incompletely. See Unable to resuspend the RNA
pellet after precipitation in Troubleshooting
Too great a mass of cultured cells is processed in a limiting volume, increasing the
viscosity of the lytic mixture and causing significant loss of RNA to the phenol
phase or the interphase layer
Decrease the viscosity of the solution by passing the sample through an
18–21 gauge needle several times
DNA contamination
The lytic solution is too viscous and some DNA is left in the aqueous layer. To
reduce and eliminate viscosity, see the recommendations in The yield of RNA is
lower than expected in Troubleshooting
Some of the interphase layer was removed with the aqueous layer. See Interphase
layer is not well resolved in Troubleshooting
See Appendix V: Avoiding DNA Contamination
PCR amplification of cDNA made
from isolated RNA is smeary with
excessive background bands
RNA Isolation Kit
Use a smaller quantity of RNA as a template for cDNA (for 1 μg of total RNA
reverse-transcribed into cDNA in a 50-μl volume, 1/5 of the cDNA reaction is
enough to amplify extremely rare messages)
9
APPENDIX I: SPECTROPHOTOMETRIC QUANTITATION OF RNA
Note
1.
Accurate spectrophotometric measurement of RNA in a 500-μl
cuvette requires ≥1 μg of RNA. Measurements that register less
than optical density 0.05 (OD260) are unacceptable.
Zero the spectrophotometer at 260 nm with DEPC-treated water,
5 mM Tris-HCl (pH 7.5), or TE buffer.
Note
2.
If the DEPC-treated water has pH<7, the quantitation should
be performed in 5 mM Tris-HCl (pH 7.5) or TE buffer (see
Preparation of Media and Reagents). The low pH will alter
the OD measurements between 260 and 280 nm, indicating a
low purity.
If using a 500-μl cuvette, place 5 μl of the RNA solution into 495 μl of
the diluent. Place a piece of laboratory film (e.g., Parafilm® laboratory
film) over the top of the cuvette and mix the sample well. Take the
spectrophotometric reading. The conversion factor for RNA is
0.040 μg/μl per OD260 unit. For a reading of 0.10, calculate the
concentration as follows:
(Spec. reading A260) × (Dilution factor) × (Conversion factor A260) =
Final concentration
Example
10
(0.10) × (500/ 5) × (0.040mg / ml) = 0.4mg / ml
3.
Calculate the yield of RNA by multiplying the volume in microliters by
the concentration. For example, in the sample above, a volume of
100 μl results in a yield of 40 μg.
4.
Re-zero the spectrophotometer with the desired solution at 280 nm.
Calculate the purity of the RNA by measuring the OD at 280 nm. The
ratio of the 260-nm measurement to the 280-nm measurement indicates
purity. Ratios of 1.8–2.0 are very pure. Lower ratios indicate protein
contamination or low pH in the solution used as a diluent for the
spectrophotometric readings.
RNA Isolation Kit
APPENDIX II: FORMALDEHYDE GEL PROTOCOL
Preprotocol Considerations
Caution Formaldehyde is a suspected carcinogen and must be used and
disposed of in accordance with federal, state, and local
regulations. Always use formaldehyde in a fume hood.
The secondary structure of mRNA present in the total RNA must be
denatured if the molecules are to migrate at their true molecular weights.
The percentage of agarose used affects resolution and transfer. High agarose
concentrations improve resolution but decrease the rate and efficiency of
RNA transfer to membranes. For large transcripts (>3500 bases), the agarose
concentration should not exceed 0.8%. The following protocol is
recommended for RNA of most sizes.
Protocol
1.
Lyophilize the RNA samples without heat until the samples are dry.
For most applications, 5–15 μg of RNA works well. More than 15 μg
of RNA may cause the lanes to become distorted with ribosomal RNA.
Note
RNA Isolation Kit
The RNA can be dried completely without subsequent
problem with resuspension since the loading buffer contains
48% formamide.
2.
In a flask, melt 1 g of agarose in 85 ml of deionized water.
3.
Add 10 ml of 10× MOPS buffer (see Preparation of Media and
Reagents) to the agarose solution. Allow the gel solution in the flask to
cool to 55°C while preparing an electrophoresis gel mold. Place the gel
mold on a level space inside a fume hood. Add 5.4 ml of 37%
formaldehyde to the cooled agarose. Swirl the flask to mix the contents
and quickly pour the agarose into the gel mold. If you wish to transfer
the RNA on the gel to a membrane, the gel should only be thick enough
to handle easily (0.5–0.75 cm). Insert the comb, and allow the gel to
solidify in the fume hood.
11
4.
While the gel solidifies, bring a water bath to boiling and prepare
5 μl of sample loading buffer for each sample. No more than 12 hours
before use, prepare the loading buffer by mixing the components listed
below:
For 100 μl total volume of loading buffer use:
48 μl of deionized formamide
17.3 μl 37% formaldehyde solution
34.7 μl loading dye (see Preparation of Media and Reagents)
Note
The loading buffer is not stable. Do not use the buffer more
than 12 hours after preparation.
5.
Cover the solidified gel with 1× MOPS buffer. Carefully pull the comb
out and connect the electrophoresis apparatus to a power supply.
6.
Resuspend the lyophilized RNA in 5 μl of loading buffer. Boil the
sample for 2 minutes, centrifuge the sample to collect condensation,
and immediately load the sample onto the gel.
7.
Electrophorese the gel at 100 V. Ethidium bromide in the loading dye
will migrate to the negative electrode, and the bromophenol blue will
travel to the positive electrode with the RNA sample. Run the
bromophenol blue from one-half to three-quarters of the length of the
gel (depending on the desired resolution).
Note
8.
Formaldehyde gels are more fragile than other agarose gels.
Use caution when moving the gel. Wear UV-protective safety
glasses or a full safety mask to prevent UV damage to the
face and skin.
Examine the gel under UV illumination.
Expected Results
The majority of eukaryotic mRNA falls within the size range of
400–2000 bases. If a size marker is unavailable, the upper and lower
ribosomal RNA bands can be used to help size the RNA. The large 28s band
is ~5 kb, and the smaller 18s band is ~2 kb. These numbers are only
approximate, since ribosomal RNA sizes vary between species.
If proceeding with northern blotting, the gel should be photographed
alongside a ruler, with the zero point of the ruler placed at the wells. Do not
allow the surface of the gel to become dry prior to transfer.
12
RNA Isolation Kit
APPENDIX III: NORTHERN TRANSFER PROTOCOL
Preprotocol Considerations
We recommend using systems that transfer RNA before the gel matrix
collapses. The protocols in this manual for formaldehyde gels and northern
transfer work well because RNA is loaded in a small volume and
immediately sinks to the bottom of the well. As RNA migrates, it stays in
the bottom of the gel. When the gel is blotted, as described below, the gel is
placed bottom side up, which requires the RNA to migrate only a short
distance before reaching the membrane.
Alternatively, the RNA may be transferred overnight to a solid support
through capillary transfer. In this system, a wick continues to supply the gel
with buffer as the light pressure of absorbent materials creates a slow
capillary flow.
Protocol
1. If the mRNA of interest is >2.5 kb, pretreat the gel by soaking in
0.05 M NaOH for 20–30 minutes. (The alkaline conditions partially
hydrolyze the mRNA, improving transfer efficiency.) Follow the alkali
soak with a 30–minute neutralization solution containing
0.1 M Tris-HCl (pH 7.5) and 0.15 M NaCl.
2. Prepare 500 ml of 10× SSC (see Preparation of Media and Reagents).
Place a solid support, such as a glass plate, over a container on a level
surface. Cut a strip of Whatman® 3MM paper long enough to bridge the
support and reach the bottom of the container on both sides (see
Figure 1).
3. Wet the Whatman 3MM paper with 10× SSC and drape the paper over
the support. While wearing gloves, remove any air bubbles trapped
between the paper and the glass.
4. Place the gel, well side down, on the Whatman 3MM paper. Remove
any bubbles trapped between the gel and the paper.
5. Cover the exposed areas of the Whatman 3MM paper that surround the
gel with laboratory film (e.g., Parafilm laboratory film) or plastic wrap.
Note
RNA Isolation Kit
This prevents the absorbent material placed on top of the gel
from "short-circuiting" the capillary transfer by coming into
contact with wet material. The only connection between the
10× SSC reservoir and the absorbent material should be
through the gel.
13
6. Cut out a piece of membrane and two pieces of Whatman 3MM paper
that are the size of the gel or slightly bigger. On the area of the
membrane to be placed above the wells, write an identifying label and
any information which will assist you in recalling the orientation of the
loaded samples.
Note
Nitrocellulose membranes work well for sensitivity but are
relatively fragile compared to nylon membranes. If the
northern blot is to be stripped and reprobed, nylon membrane
should be used.
7. Wet the membrane in water, then in 10× SSC. Place the membrane,
writing side down, on top of the gel. Remove any bubbles between the
membrane and the gel.
8. Wet the two pieces of Whatman 3MM paper in 10× SSC and place
them on top of the membrane. Remove any trapped bubbles.
9. Place 8–10 cm of absorbent material (paper towels or blotting material)
on top of the Whatman 3MM paper and place a weight and a support
(not exceeding 800 g in weight) on top of the absorbent material. Check
to see that the support is balanced by using a level. A balanced support
ensures equal blotting over the surface of the gel and prevents the
assembly from toppling over.
10. Check the level again after about 30 minutes because the absorbent
material sometimes settles unevenly.
11. Allow the transfer to proceed for 12–18 hours.
12. Remove the absorbent material. Do not remove the top two sheets of
Whatman 3MM paper. Remove the laboratory film or plastic wrap and
cut the Whatman 3MM paper underneath, leaving a border of
approximately 2 cm around the gel.
13. Without disturbing any of the intervening components, turn the entire
pile, including the bottom sheet of Whatman 3MM paper, upside down.
Place the pile on a level surface and gently peel off the Whatman 3MM
paper which was on the bottom (now on top).
14 Trace the well locations with a sharp pencil by putting the pencil tip
through the wells and marking the membrane (now under the gel).
15. Carefully remove the agarose gel.
16. Check the transfer efficiency by staining the gel in ethidium bromide
and examining the gel under UV illumination (or shine a hand-held UV
lamp directly on the damp membrane).
Caution Wear UV-protective safety glasses or a full safety mask to
prevent UV damage to the face and skin.
14
RNA Isolation Kit
17. The image of the UV-illuminated membrane can now be photographed.
Note
Limit exposure to UV light if the RNA is to be UV-fixed to the
membrane, since over-irradiation can cause a decrease in
hybridization signal.
18. Fix the RNA to the membrane permanently by UV-crosslinking or
baking under vacuum for 2 hours at 80°C.
Store the dry membrane in plastic wrap or a heat-sealable bag until the
hybridization procedure.
FIGURE 1 Capillary northern blot setup.
RNA Isolation Kit
15
APPENDIX IV: HYBRIDIZATION PROTOCOL
Preprotocol Considerations
Greater signal can be detected in less time when the hybridization volume is
minimized and the probe has a very high specific activity. Randomly primed
probes, such as those produced by the Prime-It II random primed labeling
kits and hybridization solutions, which contain volume excluders, work very
well in this application. If using a single-stranded DNA or RNA probe,
ensure that the probe is complementary, not homologous, to the target
mRNA.
Probing with nonradioactive probes can be difficult with northern blots
because the blocking buffers used for the biotin–streptavidin–alkaline
phosphatase systems often contain significant amounts of RNase, which can
degrade the RNA present on the blot. The Stratagene Illuminator
nonradioactive detection system is a chemiluminescent probe system for
northern blots that can detect RNA messages with sensitivity equivalent to
radioactive probes.
Protocol
1. Wet the membrane in deionized water. Gently shake or blot the
membrane on Whatman paper to remove most of the water.
2. Add the membrane to a container containing an appropriate amount of
Stratagene QuikHyb rapid hybridization solution or an appropriate
amount of 6× SSC buffer, 2× Denhardt's reagent, and 0.1% SDS. Cover
the membrane completely with the solution.
3. Prehybridize by shaking or rotating the container with the blot at 68°C
for 30 minutes with QuikHyb solution or for 2 hours with a standard
prehybridization solution.
4. Hybridize at a temperature consistent with the probe length and
identity. For oligonucleotide probes, the hybridization temperature
should be 5–10°C below the melting temperature (Tm). Use the
following equations to calculate the melting temperature:
OLIGONUCLEOTIDES SHORTER THAN 18 BASES
Tm = 2°C(A + T) + 4°C(G + C)
OLIGONUCLEOTIDES 14 BASES AND LONGER (MAXIMUM OF 60–70 NUCLEOTIDES)
Tm = 81.5 + 16.6(log10[Na+]) + 0.41(% G + C) – (600/N)
where N is the chain length.
For probes with perfect homology longer than 100 bases, hybridization
at 68°C works well. For smaller probes or probes with imperfect
matches to the target sequence, hybridize at 45–50°C and use the
washing step below (step 8) to remove nonspecific hybridization.
5. Denature the probe by boiling for 5 minutes.
16
RNA Isolation Kit
6. Centrifuge the sample to collect condensation, and add the sample to
the prehybridizing blot at a concentration of 1–2 × 106 cpm/ml of
hybridization solution.
7. Hybridize for 1 hour with QuikHyb solution or for 16–24 hours with
another hybridization solution.
8. If the probe is short or imperfectly matched to the target, wash the blot
four times in 2× SSC buffer and 0.1% SDS at 45°C. Otherwise, wash
the blot in four 15-minute rinses of 0.1× SSC buffer and 0.1% SDS at
60°C.
9. Wrap the membrane in plastic wrap and expose the membrane to X-ray
film at –70°C with an intensifying screen for 24–48 hours. Do not let
the blot dry.
Notes
If the blot is to be stripped and reprobed, or washed at higher
temperatures, keep the membrane damp. After the membrane
dries, the probe can become permanently affixed to the
membrane. For best results, seal the wet membrane in a
heat-sealed bag.
After the autoradiograph is developed, if significant
nonspecific signal is still present, wash the blot at gradually
higher temperatures and lower SSC concentrations until the
nonspecific signal is gone. Small probes or probes made from
degraded template may bind in significant amounts to the
ribosomal RNA bands.
RNA Isolation Kit
17
APPENDIX V: AVOIDING DNA CONTAMINATION
Genomic DNA contamination of RNA isolated with the RNA Isolation Kit
is minimal and should not cause a problem in most PCR amplifications.
PCR amplification is so powerful, however, that contamination, by even a
few molecules of DNA, can sometimes be detected. If genomic DNA
contamination is a problem with the templates and primers of choice, one of
the following strategies might help:
♦ Choose primer sets that amplify the processed mRNA across the splice
junctions, making the unprocessed genomic DNA an unamplifiable
target.
♦ Choose primer sets that amplify cDNA made from processed mRNA
and not made from an unprocessed genomic DNA template, which
would be too large to PCR-amplify effectively.
♦ Choose primer sets to create genomic DNA and cDNA amplification
products that are different sizes.
♦ Treat the RNA with RNase-free DNase I according to the following
protocol:
18
1.
Assemble the following reaction in an RNase-free tube:
1–5 μg of total RNA
1 μl of 10× DNase I buffer (see Preparation of Media and
Reagents)
1 U of DNase I (RNase-free)
Bring the volume to 10 μl with DEPC-treated water
2.
Incubate the reaction for 15 minutes at room temperature.
3.
Add 1 μl of RNase-free 20 mM EDTA to the solution and heat the
tube at 65°C for 10 minutes.
RNA Isolation Kit
PREPARATION OF MEDIA AND REAGENTS
Notes
Prepare all solution with DEPC-treated water. Use a 0.1% (v/v) solution of DEPC in
distilled water (dH2O). Allow the DEPC-treated water to incubate overnight at room
temperature and then autoclave the DEPC-treated water prior to use.
If a solution contains Tris base, prepare the solution with DEPC-treated water and
autoclave the solution before adding Tris base. Autoclave the solution once again after the
addition of Tris base to the solution.
Denaturing Solution
4 M guanidine isothiocyanate (GITC)
0.02 M sodium citrate
0.5% sarcosyl
10× DNase I Buffer
200 mM Tris-HCl (pH 8.3)
500 mM KCl
25 mM MgCl2
1 mg/ml nuclease-free bovine serum
albumin (BSA)
Loading Dye
160 μl 10× MOPS Buffer
100 μl DEPC-treated water
100 μl ethidium bromide (10 mg/ml)ll
80 μl sterile glycerol
80 μl saturated bromophenol blue in sterile water
Note Loading dye is stable if kept in a light-proof
container. Always mix well before using. To
make saturated bromophenol blue, add a
small amount of bromophenol blue crystals
to a microcentrifuge tube and vortex.
Centrifuge the sample briefly and look for
the presence of a pellet. Do not disturb the
pellet when transferring the solution to add
to the loading dye.
ll
10× MOPS Buffer
0.2 M MOPS
(3-[N-morpholino]propanesulfonic
acid)
0.05 M sodium acetate
0.01 M EDTA
Bring to a final pH of 5.5–7.0 with NaOH.
Do not autoclave
If the RNA is to be transferred to a solid support
(northern blot), use 5 mg/ml ethidium
bromide solution.
20× SSC Buffer (per Liter)
175.3 g of NaCl
88.2 g of sodium citrate
800.0 ml of water
Adjust to pH 7.0 with a few drops of
10.0 N NaOH
Adjust volume to 1 liter with water
TE Buffer
5 mM Tris-HCl (pH 7.5)
1 mM EDTA
RNA Isolation Kit
19
REFERENCES
1.
2.
3.
Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J. (1979)
Biochemistry 18(24):5294-9.
Chomczynski, P. and Sacchi, N. (1987) Anal Biochem 162(1):156-9.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
ENDNOTES
Parafilm® is a registered trademark of the American National Can.
Polytron® is a registered trademark of Kinematica AG.
Teflon® is a registered trademark of E.I. du Pont de Nemours & Co., Inc.
Whatman® is a registered trademark of Whatman Ltd.
MSDS INFORMATION
The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at
http://www.genomics.agilent.com. MSDS documents are not included with product shipments.
20
RNA Isolation Kit
RNA Isolation Kit
Catalog #200345
QUICK-REFERENCE PROTOCOL
Homogenizing Animal Tissue
♦
Prepare solution D by adding 100 μl of β-mercaptoethanol to 14 ml of denaturing solution
♦
Quickly weigh 1 g of tissue and place the tissue sample in 10 ml of solution D
♦
Homogenize the tissue in 10 ml of solution D in a glass–Teflon® homogenizer or a rotating
blade homogenizer
♦
Proceed with Isolating RNA or the Alternative Time-Saving Protocol for Isolating RNA
Preparing Tissue Culture Cells Grown in Suspension
♦
Prepare solution D by adding 100 μl of β-mercaptoethanol to 14 ml of denaturing solution
♦
Gently pellet the cells by centrifugation and discard the supernatant
♦
Add 10 ml of solution D to the cell suspension and mix thoroughly
♦
Incubate the suspension for 1 minute at room temperature
♦
Transfer the cell suspension to a 50-ml polypropylene tube and proceed with Isolating RNA
or the Alternative Time-Saving Protocol for Isolating RNA
Preparing Tissue Culture Cells Grown in Monolayer
21
♦
Prepare solution D by adding 100 μl of β-mercaptoethanol to 14 ml of denaturing solution
♦
Decant the tissue culture medium from the tissue culture plates
♦
Add 10 ml of solution D to a tissue culture plate and swirl the plate gently for 30 seconds
♦
Transfer solution D from the tissue culture plate to a second tissue culture plate and swirl the
second plate for 30 seconds. Repeat this step for the remaining plates.
♦
Transfer the cell–solution D mixture to a 50-ml polypropylene tube and proceed with
Isolating RNA or the Alternative Time-Saving Protocol for Isolating RNA
Isolating RNA
♦
Add 1.0 ml of 2 M sodium acetate (pH=4.0) to the tube and mix by inversion
♦
Add 10.0 ml of phenol (pH 5.3-5.7) to the tube and mix by inversion
♦
Add 2.0 ml of chloroform–isoamyl alcohol to the tube. Cap the tube tightly and shake the
tube vigorously for 10 seconds
♦
Incubate the tube on ice for 15 minutes
♦
Transfer the sample to a prechilled, 50-ml, thick-walled, round-bottom centrifuge tube and
spin in a centrifuge at 10,000 × g for 20 minutes at 4°C
♦
Transfer the aqueous phase, which contains the RNA, to a fresh centrifuge tube
♦
22
Add an equal volume of isopropanol to the tube and incubate the tube for ≥1 hour at –20°C
to precipitate the RNA
♦
Spin the tube in a centrifuge at 10,000 × g for 20 minutes at 4°C
♦
Dissolve the pellet, which contains the RNA, in 3.0 ml of solution D
♦
Add 3.0 ml of isopropanol to the tube and incubate the tube for 1 hour at –20°C
♦
Spin the tube in a centrifuge at 10,000 × g for 10 minutes at 4°C. Discard the supernatant
♦
If the salt concentration is important, wash the pellet with 75% (v/v) ethanol
♦
Discard the supernatant and dry the pellet under vacuum for 2–5 minutes
♦
Resuspend the RNA in 0.5–2 ml of DEPC-treated water at an A260/A280 ratio ≥1.8
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