Separation of RNA in
Agarose Gels

Section VIII: Separation of RNA in Agarose Gels

In This Section
Introduction
Preparation of RNA Samples
Buffers for RNA Electrophoresis
Electrophoresis of RNA
Detection of RNA in Agarose Gels with
GelStar® or SYBR® Green II Gel Stains
Detecting RNA with Ethidium Bromide
Nothern Blotting
FlashGel® System for RNA Analysis
References

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Section VIII: Separation of RNA in Agarose Gels

Introduction
Separation of RNA in agarose gels is used for a numberof different purposes, including Northern blots to monitor
RNA expression levels, checking RNA integrity and sizeselection of RNA for cloning experiments. Separation of
RNA based on fragment length requires conditions thatare different from DNA analysis. These include sample
preparation, the use of sample and gel denaturants,
electrophoresis buffers, and visualization.
Denaturing systems
The purpose of the experiment and the size of the RNA
being separated are the primary drivers in determining
which denaturing system to use. The most frequently
used denaturants for RNA agarose gel electrophoresis
are formaldehyde, formaldehyde/formamide, and glyoxal
plus DMSO. In each system, the denatured RNA migrates
through the agarose gel in a linear relation to the log of
its molecular weight (similar to DNA). The most efficient
RNA denaturant is methylmercury hydroxide. Because of
the hazards associated with this denaturant, it is the least
used system for RNA analysis.

The resolving powers of the glyoxal/DMSO and theformaldehyde buffer systems are nearly identical. For
detection by Northern analysis, glyoxal/DMSO denaturant
is preferable because these gels tend to produce sharperbands than the formaldehyde system. Glyoxal gels require
more care to run than formaldehyde gels and because of
the lower buffering capacity of glyoxal, these gels must be
run at lower voltages than gels containing formaldehyde.
Glyoxal gels require a phosphate electrophoresis buffer
and the buffer must be recirculated during electrophoresis.
If the pH of the buffer rises above 8.0, glyoxal dissociatesfrom RNA, causing the RNA to renature and migrate in an
unpredictable manner.
For staining purposes, either denaturant can be used.
Ethidium bromide, GelStar® Nucleic Acid Gel Stain and
SYBR® Green II Gel Stain bind formaldehyde-denatured
RNA more efficiently than glyoxal-denatured RNA. Glyoxal
denaturant can interfere with binding of the stain, but
gel backgrounds are often lower in these gels than withformaldehyde-denatured gels.
It is important to minimize RNase activity when runningagarose gels by following certain precautions. There areseveral agents on the market that effectively remove
RNase’s or consult Sambrook, et al.

Section VIII: Separation of RNA in Agarose Gels
Preparation of RNA Samples

Separation of RNA in
Agarose Gels

Which sample denaturation method to choose depends
on the final goal of the experiment and the secondary
structure of the RNA. There are several procedures tochoose from, the most useful of which are described here.
Any sample denaturation method can be used with any of
the gel buffering systems. If simply checking the integrityof cellular RNA, no sample denaturation is necessary andTAE or TBE Buffer can be used.
Common denaturants
Formamide
Formaldehyde
GlyoxalApplication
Retain biological activity
Sample recovery
Northern blotting;significant secondary
structure

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Formamide-only denaturation
Formamide denaturation is suitable for almost all RNA
samples and is recommended if you need to retain
biological activity. Gels can be cast and run in standard
TAE or TBE Buffer Systems or MOPS Buffer. If there isa significant amount of secondary structure, anothersample denaturation method should be chosen.
■■ Materials–– Water bath set to 70°C–– Ice■■ Reagents–– AccuGENE® Molecular Biology Water (RNase-free)–– Deionized formamide–– AccuGENE® 10X MOPS Buffer(200 mM MOPS, pH 7.0, 50 mM sodium acetate,
10 mM EDTA, 10 mM EGTA), pH 7.0

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Section VIII: Separation of RNA in Agarose Gels
Preparation of RNA Samples — continued

Formamide denaturation of RNA samplesB ring the RNA volume up to 8 µl with RNase-freewater.
Add 2 µl of 10X MOPS Buffer.
Add 9 µl of deionized formamide.
M ix thoroughly.
Heat at 70°C for 10 minutes.
Chill on ice for at least 1 minute before loading.

1.
2.
3.
4.
5.
6.

Formaldehyde denaturation
Formaldehyde denaturation is suitable whensamples are to be recovered. It is necessary toensure that the formaldehyde is fully removed fromthe recovered RNA prior to subsequent studies.
Some enzymatic reactions, such as in vitro transcription,
may be problematic even after complete removal of theformaldehyde.

Glyoxal denaturation
Glyoxal is a very efficient denaturant, but should not be
used if samples are to be recovered. Glyoxal denatures
RNA by introducing an additional ring into the guanosine
residues, thus interfering with G-C base pairing. Glyoxal
denaturation would be the recommended procedure forNorthern blotting. Typically a phosphate electrophoresis
buffer is recommended with recirculation to prevent
formation of a pH gradient. Alternatively, a 10 mM PIPES,
30 mM bis-Tris buffer, or a 20 mM MOPS, 5 mM sodium
acetate, 1 mM EDTA, 1 mM EGTA Buffer systems can also
be used without recirculation.
■■ Materials–– Water bath set to 50°C–– Ice■■ Reagents■■ Materials–– AccuGENE® Molecular Biology Water (RNase-free)–– Water bath set to 70°C–– DMSO–– Ice–– 100 mM Sodium phosphate, pH 7.0■■ Reagents–– 	Mix 5.77 ml of 1 M Na HPO with 4.23 ml of 1 M
NaH PO
2–– AccuGENE® Molecular Biology Water (RNase-free)–– 	Adjust volume to 100 ml with RNase-free water–– AccuGENE® 10X MOPS Buffer(200 mM MOPS, pH 7.0, 50 mM sodium acetate,
10 mM EDTA, 10 mM EGTA), p H 7.0–– 6 M Glyoxal, 40% (v/v) solution, deionized
immediately before use–– 37% (v/v) formaldehyde–– 	Pass solution through a small column of
mixed-bed ion exchange resin until the pH is >5.0;
large volumes can be deionized then stored
frozen in aliquots at –20°C–– Deionized formamide
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Formaldehyde denaturation of RNA samples
1.
2.
3.
4.
5.
6.

B
 ring the RNA volume up to 6 µl with RNase-freewater.
Add 2 µl of 10X MOPS Buffer.
Add 2 µl of 37% formaldehyde.
A dd 9 µl deionized formamide.
Mix thoroughly.
Heat at 70°C for 10 minutes. Chill on ice for atleast 1 minute before loading.

42

Separation of RNA in
Agarose Gels

Returnto MainGlyoxal denaturation of RNA samples
1.
2.
3.
4.
5.
6.

B
 ring the RNA volume up to 11 µl with RNasefree water
Add 4.5 µl of 100 mM sodium phosphate.
Add 22.5 µl of DMSO
A dd 6.6 µl of deionized glyoxal.
Mix thoroughlyHeat at 50°C for 1 hour. Chill on ice for at least
1 minute before loading

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Section VIII: Separation of RNA in Agarose Gels
Buffers for RNA Electrophoresis

The two commonly used buffer systems for RNA
electrophoresis are a phosphate buffer for glyoxal/DMSO
denatured RNA and a MOPS Buffer for formaldehyde orformamide denatured RNA. These buffers are very low
in ionic strength. During electrophoresis, a pH gradient
may be generated along the length of the gel, resulting in
the hydrolysis (melting) of the agarose gel. This problem
can be avoided by recirculating the buffer. For glyoxaldenatured gels, if the pH of the buffer rises above pH 8.0,
the glyoxal will dissociate from the RNA, causing the RNA
to renature and migrate in an unpredictable manner.

Buffer preparation
Note: Use RNase-free chemicals, water and containers
100 mM Sodium Phosphate Buffer, pH 7.0(Glyoxal/DMSO denatured RNA)

1 M Na2HPO4
1 M NaH2PO4

g/l
57.7 ml42.3 ml—— Adjust volume to 1 liter with RNase-free water—— Adjust volumes accordingly to prepare more buffer
10X MOPS Buffer(Formaldehyde or Formamide denatured RNA)

200 mM MOPS (free acid)
50 mM sodium acetate
10 mM EDTA•2H2O
10 mM EGTA (free acid)g/l
41.86 g6.80 g3.72 g3.80 g—— Mix with 850 ml of distilled water—— Adjust pH to 7.0 with 10 M NaOH—— Adjust volume to 1 liter with RNase-free water—— Filter through a 0.2 µm nitrocellulose filter and store
in the dark
Caution: NaOH is s corrosive material. Use safety
glasses and gloves to protect from burns.

Separation of RNA in
Agarose Gels

Section VIII: Separation of RNA in Agarose Gels
Electrophoresis of RNA

The choice of an agarose free of RNase contamination isof major importance. Lonza offers a variety of agarose
products for RNA electrophoresis including The FlashGel®
System for RNA, Reliant® and Latitude® Precast RNA Gels,
Lonza agarose, AccuGENE® 10X MOPS Buffer, RNA Marker,
GelStar® and SYBR® Green Nucleic Acid Gel Stains.

General guidelines—— Northern blotting requires a standard melting
temperature agarose such as SeaKem® LE or
NuSieve® 3:1 Agarose or Reliant® or Latitude® Precast
RNA Gels—— If samples are to be recovered, a low melting
temperature agarose can be used such as NuSieve®
GTG® or SeaPlaque® GTG® Agarose—— A 1.5 - 2.0% gel made with SeaKem® GTG® or SeaKem®
Gold Agarose or FlashGel® System, Reliant® or
Latitude® Precast RNA Gels will work for RNA moleculesof 500 - 10,000 nucleotides

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Section VIII: Separation of RNA in Agarose Gels
Electrophoresis of RNA — continued—— For RNA smaller than 500 nucleotides, use a 3 or 4%
NuSieve® 3:1 or MetaPhor® Agarose Gel—— For RNA larger than 10,000 nucleotides, SeaKem® Gold
Agarose and FlashGel® System, Reliant® or Latitude®
Precast RNA Gels will be a better choice for tighterbands and better resolution—— If a low melting temperature agarose is required,a 1.5 or 2.0% SeaPlaque® GTG® Gel can be used forseparation of RNA from 500 - 10,000 nucleotides, while
a 3.0% or 4.0% NuSieve® GTG® Gel should be used forfine resolution of RNA smaller than 500 nucleotides;
NuSieve® GTG® Agarose is not recommended forformaldehyde/MOPS gels

Dye mobilities for RNA gels
The following table is a migration table of single-stranded
RNA in relation to bromophenol blue (BPB) and xylene
cyanol (XC) in formaldehyde or glyoxal agarose gels.
Formaldehyde gels Glyoxal gels
Agarose (% w/v) 		 XC BPB
XC
BPB
SeaKem® Gold Agarose
1.0

6,300 660

9,500

940

1.5

2,700 310

4,300

520

2.0
1,500 200
2,300
SeaKem® GTG® and SeaKem® LE Agarose

300

1.0

4,200 320

7,200

740

1.5

1,700 140

2,800

370

2.0
820 601,600
SeaPlaque® and SeaPlaque® GTG® Agarose

220

1.0

2,400 240

4,400

400

1.5

800

80

1,900

180

2.0
490
NuSieve® 3:1 Agarose

30

1,050

120

2.0

950

70

1,600

155

3.0

370

20

740

75

4.0
190 5
370
NuSieve® GTG® and MetaPhor® Agarose

40

2.0

Not applicable

1,300

150

3.0

Not applicable

480

60

4.0

Not applicable

260

40

Separation of RNA in
Agarose Gels

Returnto MainAgarose selection guide for RNA electrophoresis

Separation		

Northern Blotting

Preparative

Fragments<500 bases

NuSieve® 3:1 Agarose
MetaPhor® Agarose

NuSieve® 3:1 Agarose

NuSieve® GTG® Agarose

Fragments between
500-10,000 bases

SeaKem® LE Agarose		
SeaKem® GTG® Agarose

SeaKem® LE Agarose
SeaKem® GTG® Agarose

SeaPlaque® GTG® Agarose

Fragments >10,000
bases

SeaKem® Gold Agarose

SeaKem® Gold Agarose

SeaPlaque® GTG® Agarose

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Section VIII: Separation of RNA in Agarose Gels
Electrophoresis of RNA — continued

Electrophoresis of gels containing formaldehyde
A formaldehyde denaturant with a MOPS buffer is themost commonly used system for RNA electrophoresis.
Care should be taken when handling gels containing
formaldehyde. These gels are less rigid than other agarose
gels. NuSieve® GTG® Agarose is not compatible with the
MOPS buffering system.
Formaldehyde electrophoresis of RNA
Note: Do not exceed 20 µg of RNA per lane as larger

amounts can result in loss of resolution.
1.

For a 1% gel, dissolve 1.0 g of agarose in 72 ml of water.

■■ Materials–– Water bath set to 60°C–– Fume hood–– Accessories to cast an agarose gel–– Electrophoresis chamber and power supply–– Flask or beaker■■ Reagents–– AccuGENE® 10X MOPS Buffer prewarmed to 60ºC–– 1X MOPS Buffer 37% (v/v), formaldehyde
prewarmed to 60°C

2.

Cool agarose to 60°C in hot water bath–– Formaldehyde Loading Buffer (1 mM EDTA, pH 8.0,
0.4% bromophenol blue and xylene cyanol,
50% glycerol)3.

Place in fume hood–– Distilled water4.

I mmediately add 10 ml of prewarmed 10X MOPS Buffer5.

Add 5.5 ml of prewarmed 37% formaldehyde

6.

Cast gel in a fume hood7.

D enature the RNA sample following one of the methods
previously described

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

8.

A dd 2 µl of formaldehyde loading buffer per 20 µl ofsample9.

Mix thoroughly

10.

Remove the gel comb

11.

Place the gel in the electrophoresis chamber

12.

C over surface of the gel to a depth of 1 mm with 1X MOPS
Buffer13.

L oad the samples

14.

E lectrophorese at a maximum of 5 V/cm (interelectrodedistance) until the bromophenol blue has traveled atleast 80% of the way through the gel (see mobility tableon page 135)

Adjust the amounts for different percent gels

Separation of RNA in
Agarose Gels
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Section VIII: Separation of RNA in Agarose Gels
Electrophoresis of RNA — continued

Electrophoresis of gels containing glyoxal/DMSO■■ Materials

These gels should be run slower than formaldehyde gelswith buffer recirculation to avoid the formation of a pH
gradient. Glyoxylated RNA will give sharper bands thanformaldehyde-treated RNA.

–– Water bath set to 60°C

Glyoxal/DMSO electrophoresis of RNA–– Flask or beaker

1.

For a 1% gel, dissolve 1.0 g of agarose in 100 ml of 10 mM

sodium phosphate, pH 7.0. Adjust amounts for different
percent gels.
2.

Cool agarose to 60°C in hot water bath.

3.

Cast the gels to a thickness that will accommodate aloading volume of 60 µl.
4.

Remove the comb.

5.

Place the gel in the electrophoresis chamber.

6.

Cover gel with 10 mM sodium phosphate buffer to a depth of

1 mm.
7.

Add 12 µl of glyoxal loading buffer per 45 µl of sample.

8.

Mix thoroughly.

9.

Load 0.5 µg - 1.0 µg of RNA per lane.

10.

Electrophorese at 4 V/cm (interelectrode distance)–– Fume hood–– Accessories to cast an agarose gel–– Electrophoresis chamber and power supply–– Recirculating unit■■ Reagents–– 100 mM sodium phosphate pH 7.0–– Glyoxal loading buffer(10 mM sodium phosphate, pH 7.0, 0.25%
bromophenol blue and xylene cyanol, 50% glycerol)
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

while the buffer is recirculated. If no recirculation
apparatus is available, pause electrophoresis every 30
minutes and remix the buffer.
11.

Electrophorese until the bromophenol blue has traveled
at least 80% of the way through the gel (see mobility
table on page 135).

Separation of RNA in
Agarose Gels

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Section VIII: Separation of RNA in Agarose Gels
Detection of RNA with GelStar® or SYBR® Green II Gel Stains

Introduction
Detection of RNA in agarose gels varies depending on
the denaturant, stain and photographic conditions used.
Lonza offers two highly sensitive stains, GelStar® andSYBR® Green II Gel Stains, for the detection of RNA in
agarose gels. These stains exhibit higher RNA detection
sensitivity than ethidium bromide, allowing you to load
less RNA sample on your gel. Unlike ethidium bromide,
GelStar® and SYBR® Green II Stain only fluoresce uponbinding to the nucleic acid resulting in lower background
fluorescence which is particularly useful when including
the stain in the gel for glyoxal denatured samples or whenconcentrations of formaldehyde in the gel exceed 2 M. The
chart below shows the detection sensitivity of various
dyes for RNA using in-gel or post-staining techniques.
Stain
Method Native RNA Glyoxal RNA
GelStar® Stain
in-gel10 ng
10 ng
Ethidium bromide,
no destain
in-gel30 ng
150 ngEthidium bromide,with destain
in-gel
30 ng
50 ng
SYBR®
Green II Stain
post stain 15 ng
50 ng—— A 2X working solution of GelStar® or SYBR® Green Stain
should be prepared just prior to use from the 10,000X
stock solution by diluting in 1X MOPS Buffer or a pH 7.5
to 8.5 buffer (e.g., TAE, TBE or TE).
—— For nondenaturing or denaturing polyacrylamide
gels, dilute GelStar® or SYBR® Gel Stain 1:10,000X in
1X electrophoresis buffer.
—— Agarose gels should be cast no thicker than 4 mm.
As gel thickness increases, diffusion of the stain
into the gel is decreased, lowering the efficiency ofRNA detection.
—— Optimal sensitivity for GelStar® and SYBR® Green Stains
is obtained by using the appropriate photographic
filters for each stain.
–– GelStar® Stain: Wratten® or Tiffen® #9 Filter–– SYBR® Green Stains: Wratten or Tiffen #15 Filter—— We do not recommend photographing gels with a 254
nm transilluminator. An outline of the UV light source
may appear in photographs. A filter that will allow a
525 nm transmission and exclude infrared light isrequired.

Limits are based on optimal detection methods for each stain. Samples
detected with SYBR® Green Stain were post-stained, samples detected withethidium bromide or GelStar® Stain were detected by in-gel staining (poststained gels showed similar results).

Separation of RNA in
Agarose Gels

FlashGel® System (see page 25), uses a similar stain
system and is capable of RNA detection <10ng.
Tips for staining gels with GelStar® or
SYBR® Green II Gel Stains
Follow the guidelines below to increase the detection
sensitivity of GelStar® or SYBR® Green II Gel Stains.
—— New clear polypropylene containers (e.g., Rubbermaid® Recycling #5 Plastics) should be obtained
for use with GelStar® and SYBR® Green Stains. When
stored in the dark in polypropylene containers, thediluted stain can be used for up to 24 hours and will
stain 2 to 3 gels with little decrease in sensitivity. The
containers should be rinsed with distilled water (donot use detergents) after each use and dedicated toGelStar® or SYBR® Green Stain use only—— These stains bind to glass and some non-polypropylene(polystyrene) plastics, resulting in reduced or no
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Section VIII: Separation of RNA in Agarose Gels
Detection of RNA with GelStar® or SYBR® Green II Gel Stains — Continued■■ Materials

Procedure for staining RNA with GelStar® or
SYBR® Green II Gel Stains
For optimal resolution, sharpest bands and lowest background,stain the gel with GelStar® or SYBR® Green II Gel Stain following
electrophoresis.
The photographs below demonstrate the detection
sensitivity of various stains with different sample
denaturants.
Follow the steps below to stain RNA after electrophoresis
1.

Remove the concentrated stock solution of the stain

from the freezer and allow the solution to thaw at room
temperature.
2.

Spin the solution in a microcentrifuge to collect the dyeat the bottom of the tube.
3.

Dilute the 10,000X concentrate to a 2X working solution(2 µl/10 ml) in 1X MOPS Buffer, in a clear plastic
polypropylene container. Prepare enough staining
solution to just cover the top of the gel.
4.

Remove the gel from the electrophoresis chamber.

5.

Place the gel in staining solution.

6.

Gently agitate the gel at room temperature.

7.

Stain the gel for 60 minutes.

8.

Remove the gel from the staining solution and view–– Clear polypropylene container (e.g., Rubbermaid®
Recycling #5 Plastics)–– GelStar® Photographic Filter (Wratten® #9
equivalent) or SYBR® Green Photographic Filter(Wratten® #15 equivalent)–– Microcentrifuge–– UV transilluminator Dark Reader® Transilluminator(Clare Chemical Research, Inc.) or CCD imaging
system■■ Reagents–– 1X MOPS Buffer–– GelStar® or SYBR® Green II Gel Stain stock solution
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

with a transilluminator, CCD camera or Dark Reader™
Transilluminator.

Separation of RNA in
Agarose Gels

Returnto Main

Note: Gels stained with GelStar® or SYBR® Green II Gel Stains do
not require destaining. The dyes’ fluorescence yield is muchgreater when bound to RNA than when in solution.

Ethidium bromide
A B C

GelStar® Stain
A B C

SYBR® Green I Stain SYBR® Green II Stain
A B C

A B C

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Section VIII: Separation of RNA in Agarose Gels
Detection of RNA with GelStar® or SYBR® Green II Gel Stains — continued

Follow this procedure when including GelStar® Nucleic
Acid Gel Stain in the agarose gel

GelStar®
Stain
A

B

SYBR® Green I Stain
Stain
C

A

B

C

SYBR® Green II 		
Stain
A

B

C

Note: Unlike ethidium bromide, GelStar® Nucleic Acid Gel Stain
does not interact with glyoxal.
1.

Remove the concentrated stock solution of GelStar®

Stain from the freezer and allow the solution to thaw atroom temperature.
2.

Spin the solution in a microcentrifuge tube.

3.

Prepare the agarose solution.

4.

Once the agarose solution has cooled to 70ºC, add thestain by diluting the stock 1:5,000 into the gel solution
prior to pouring the gel (2 µl per 10 ml).
5.

Slowly swirl the solution.

6.

Pour the gel into the casting tray.

7.

Load samples onto the gel .

8.

Run the gel.

9.

Remove the gel from the electrophoresis chamber.

10.

View with a 300 nm UV Transilluminator, CCD camera or

Clare Chemical’s Dark Reader® Transilluminator.
Note: GelStar® Stained Gels do not require destaining. The dye’s
fluorescence yield is much greater when bound to RNA thanwhen in solution.

Samples of E. coli total RNA were denatured using the following
denaturants: Lane A: Formaldehyde/Formamide; Lane B: Formamide;
Lane C: Glyoxal. Samples were loaded at 2 mg/lane for the formaldehyde/
formamide and formamide only denatured samples, and 4 mg/lane
for the glyoxal denatured samples. Reliant® RNA Precast Agarose Gels
were run at 7 V/cm for 40 minutes in 1X MOPS Buffer (prepared from
AccuGENE® 10X MOPS Buffer) and post-stained using GelStar® or SYBR®
Green II Stain or ethidium bromide. The left series of photographs weretaken using a Polaroid® Camera, SYBR® Green Photographic Filter on
a UV light box. The right series of photographs were taken using theSYBR® Green Photographic Filter on the Clare Chemical’s Dark Reader®
Transilluminator.

Separation of RNA in
Agarose Gels
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Section VIII: Separation of RNA in Agarose Gels
Detection of RNA with GelStar® or SYBR® Green II Gel Stains — Continued

Visualization by photography■■ Materials

Gels stained with GelStar® and SYBR® Green II Gel Stains
exhibit negligible background fluorescence, allowing long
film exposures when detecting small amounts of RNA. Use
the appropriate photographic filter for the stain you are using(see page 138).
The table below provides suggested film types andphotographic conditions
Polaroid® Film
Type 57 or 667

f-stop 	Exposure time
4.5
0.5-2 seconds

Type 55

4.5

15-45 seconds

Visualization by image capture system
GelStar® and SYBR® Green II Nucleic Acid Gel Stains arecompatible with most CCD and video imaging systems.
Due to variations in the filters for these systems, you may
need to purchase a new filter. Lonza does not sell filters forthis type of camera. Contact your systems manufacturer
and using the excitation and emission information listed,
they can guide you to an appropriate filter.

–– Staining vessel larger than the gel–– UV transillu­minator, Dark Reader® Transillu­minator(Clare Chemical Research, Inc.) or CCD imaging
system–– Magnetic stir plate–– Magnetic stir bar■■ Reagents–– Ethidium bromide stock solution (10 mg/ml)(1.0 g Ethidium bromide,100 ml distilled water,
stir on magnetic stirrer for several hours, transfer
the solution to a dark bottle, store at room
temperature)–– Distilled water–– 0.1 M ammonium acetate
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Stain 	Emission (nm) 	Excitation (nm)GelStar® Stain
527
493
SYBR® Green I Stain
521
494
SYBR® Green II Stain
513
497
Application notes—— The fluorescent characteristics of GelStar® and SYBR®
Green II Gel Stains make them compatible with argon
ion lasers

Separation of RNA in
Agarose Gels

Returnto Main—— Gels previously stained with ethidium bromide cansubsequently be stained with GelStar® or SYBR® GreenII Gel Stain following the standard protocol for poststaining. There will be some decrease in sensitivity
when compared to a gel stained with only GelStar® or
SYBR® Green II Gel Stain—— We recommend the addition of 0.1% to 0.3% SDS in the
prehybridization and hybridization solutions when
performing Northern blots on gels stained with these
dyes

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Section VIII: Separation of RNA in Agarose Gels
Detection of RNA with Ethidium Bromide

Introduction
Ethidium bromide does not stain RNA as efficiently as it
does DNA, so be certain that sufficient RNA is loaded tosee the band of interest. Although as little as 10 ng - 20
ng of DNA can usually be visualized by ethidium bromide
fluorescence, as much as 10-fold more RNA may beneeded for good visualization. Visualization of poorly
staining RNA is made even more difficult by the higher
background fluorescence of RNA gels. In formaldehydegels, background fluorescence can be minimized bydropping the formaldehyde concentration in the gel from
2.2 M to 0.66 M. Staining with ethidium bromide is notrecommended when performing Northern blot analysis
onto nylon membranes.

1.

Prepare agarose solution.

2.

While the agarose solution is cooling, add ethidium
bromide to a final concentration of 0.5 µg/ml to thesolution.

3.

Slowly swirl the solution.

4.

Pour the gel into the casting tray.

5.

Add ethidium bromide to the running buffer to a final
concentration of 0.5 µg/ml.

6.

Load and run the gel.

7.

Destain the gel by submerging the gel in distilled waterfor 60 minutes.

Prepare enough working solution of ethidium bromide

8.

Repeat in fresh distilled water.

(0.5 mg/ml of ethidium bromide in 0.1 M ammonium
acetate) to cover the surface of the agarose gel.

9.

Gels can be viewed with a UV light transilluminator, Dark
Reader® Transilluminator (Clare Chemical Research, Inc.)
or CCD imaging system.

Follow the steps below to stain RNA after electrophoresis
1.

Follow the steps below when including
ethidium bromide in the agarose gel

2.

Remove the gel from the electrophoresis chamber.

3.

Submerge the gel for 30 - 45 minutes in the ethidium

bromide solution.
4.

Remove the gel from the solution.

5.

Submerge the gel for 60 minutes in a new container

filled with distilled water.

Separation of RNA in
Agarose Gels

NOTE: For gel concentrations of 4% or greater, these times mayneed to be doubled. If after destaining the background is still
too high, continue to destain.
6.

Repeat in fresh distilled water.

7.

Gels can be viewed with a UV light transilluminator,
Dark Reader® Transilluminator (Clare Chenical Research,Inc.) or CCD imaging system.

Decontamination of GelStar® and SYBR® Green Gel Stains
and ethidium bromide solutions
Staining solutions should be disposed of by passing
through activated charcoal followed by incineration of thecharcoal. For absorption on activated charcoal, consult
Sambrook, et al., pp. 6.16 - 6.19, (1989). Follow stateand local guidelines for decontamination and disposal of
Nucleic Acid Staining Solutions.

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Section VIII: Separation of RNA in Agarose Gels
Northern Blotting

Recommended agaroses for Northern blotting

Choosing a blotting method

When transferring RNA from an agarose gel to a membrane,
a standard melting temperature agarose should be used.
Standard melting temperature agaroses have higher gel
strength than low melting temperature agaroses and do
not fracture during transfer. The table below is a list of
Lonza agaroses and precast gels that are recommended
for blotting.

Consider the following when choosing a blotting method:

AgaroseNuSieve® 3:1 Agarose
SeaKem® LE Agarose
SeaKem® GTG® Agarose
SeaKem® Gold Agarose
Reliant® & Latitude® RNA Gels

Size Range (bases)<500
500 - 10,000
500 - 10,000
500 - 10,000
25 - 10,000

Tips for agarose gel preparation—— Use the lowest agarose concentration required toresolve your fragments—— If staining with ethidium bromide prior to transfer,
thoroughly destain the gel with distilled water—— Avoid casting thick gels (>4 mm) unless absolutely
necessary. Thick gels not only require longer
electrophoretic times, but may interfere with thefree transfer of nucleic acids to the hybridization
membrane—— Do not use more than a 500.0 gm weight on top of thestack. Excess weight can compress the gel. This will
increase the agarose concentration and decrease thepore size, which can inhibit movement of the buffer
and RNA–– Gel concentration–– Fragment size–– Speed and transfer efficiency
Capillary transfer
Traditional passive capillary transfer uses paper towels to
draw the transfer buffer from a reservoir through the gel.
Passive capillary transfer takes 16 - 24 hours to complete.
The most common complaints are poor transfer efficiency
for larger RNA molecules and long transfer time.
Vacuum transfer
Vacuum transfer systems use negative pressure topull the transfer buffer through the gel along with thenucleic acid. These systems take approximately 30 - 60
minutes to complete. Vacuum blotting can be useful forhigh concentration gels where compression of the gel isa concern.
Electroblotting
Electroblotting is most often used for polyacrylamidegels. A voltage gradient pulls the sample out of the gel
and onto a membrane. Semi-dry electroblotting requires
minimal buffer with a low voltage and current. Care should
be taken so smaller fragments are not pulled completely
through the membrane.

Separation of RNA in
Agarose Gels

Returnto MainChoosing the appropriate membrane
When transferring RNA, nylon membranes have several
advantages over nitrocellulose:
Nylon
Uncharged/charged
High strength
Good for reprobing
UV crosslinking
50 nucleotides**Nitrocellulose
Supported/unsupported
Good strength*
Poor for reprobing
Baking in vacuum oven at 80ºC
500 nucleotides*** Good for supported nitrocellulose, poor for unsupported nitrocellulose.
**Lower size limit for efficient nucleic acid retention.

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Section VIII: Separation of RNA in Agarose Gels
Northern Blotting — continued

General guidelines for Northern blotting—— It is important to minimize RNase activity by following
certain precautions. There are several agents on themarket that effectively remove RNase’s or consult
Sambrook, et al—— Wear gloves throughout the procedure. RNAs are notsafe from nuclease degradation until they have been
immobilized on the membrane■■ Materials–– Nitrocellulose or nylon membrane–– 2-3 glass dishes larger than gel–– Paper towels–– Whatman® 3MM Chromatography Paper–– Glass or plastic pipette–– Flat ended forceps—— Cut or mark membrane for orientation. Do this before
wetting the membrane–– Glass plate to serve as a platform for the gel—— The blotting membrane should be in contact withthe underside of the gel. Since nucleic acids willconcentrate near the bottom of the gel, there is lessdistance for them to travel during the transfer–– <500.0 g weight—— Cut all papers and membranes to the correct size,
such that, the only thing pulling the transfer buffer isthe buffer solution—— Avoid bubbles, ensure that there is even contact
between all the layers of the blotting system—— Incorrect denaturation of probe can cause poortransfer results—— Glyoxal gels can be transferred immediately after
electrophoresis. The glyoxal will be removed in thepost transfer, prehybridization wash

Separation of RNA in
Agarose Gels—— Destain gels that contain formaldehyde or ethidium
bromide to avoid sample loss and inefficient transfer
efficiency
NOTE: Soak gel 3 times for 5 - 10 minutes each in either
1X MOPS, transfer buffer, or sterile water. This will remove
most of the formaldehyde and excess background
fluorescence seen with ethidium bromide. Complete
destaining usually takes 2 hours or longer.

–– Plastic wrap–– Orbital or rocking platform shaker■■ Reagents–– AccuGENE® Molecular Biology Water–– AccuGENE® 20X SSPE or AccuGENE® MOPS Buffer

Procedure for RNA transfer by passive capillaryelectrophoresis transfer buffer
The concentration of the transfer buffer will vary dependingon the method and type of membrane used. The capillary
method outlined here uses a nylon membrane and 5X
SSPE Buffer.
20X SSPE(3 M NaCl, 0.2 M NaH PO •H O, 0.2 M EDTA • 2H O)
175.3 gNaCl
27.6 g
NaH PO •H O
7.4 g
EDTA • 2H O
Adjust to pH 7.4 with 10 N NaOH
To 1 liter with distilled water
2

4

2

2

2

4

2

2

—— Lonza’s Reliant® and Latitude® Precast RNA Gels do notcontain any denaturants or stains and do not require
destain prior to transfer.

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Section VIII: Separation of RNA in Agarose Gels
Northern Blotting — Continued

Follow the steps below for gel preparation andsetting up the transfer■■ Materials–– Membrane

Cast a standard melting temperature agarose gel nothicker than 4 mm.

–– Plastic wrap

2.

Electrophorese RNA following standard protocols.

–– Vacuum dryer set to 80°C or hot oven at 65°C.

3.

For formaldehyde gels, follow the steps below prior totransfer. For glyoxal gels, proceed to step 4.

4.

Soak the gel in an excess of 1X MOPS Buffer, distilledwater or 5X SSPE for 10 minutes.

5.

Repeat three times with new wash solution each time.

6.

Float membrane in RNase-free water for 5 minutes.

7.

Equilibrate membrane in 5X SSPE for 5 minutes. The
membrane may remain in the transfer buffer until it is
used.

1.

8.

Set up transfer using either an upward capillary transfer
set up or downward capillary transfer set up.

9.

Allow the transfer to proceed as follows:

Transfer set-up

Time

Upward16 - 24 hours

Downward

1 - 4 hours depending on gel
thickness and concentration

Turboblotter™ System

1 - 3 hours

10.

Remove the paper toweling or chromatography paper.

11.

Remove the gel and membrane together.

12.

Mark the positions of the wells on the membrane witha pencil for orientation then remove the gel from thetransfer setup.

13.

Rinse the membrane for 30 - 60 seconds in transfer
buffer.

14.

Place the membrane on a sheet of Whatman® 3MM
Chromatography Paper.

15.

Treat membrane as described in Immobilizing RNA on amembrane, next section.

–– UV light source■■ Reagents–– AccuGENE® 1 M Tris pH 8.0–– RNase-free water–– AccuGENE® Molecular Biology Water or AccuGENE®
10X Mops Buffer–– AccuGENE® 20X SSPE
Immobilizing RNA on a membrane
The methods and procedures for immobilizing RNA on
the membrane are essentially the same as they are
for DNA.
If samples have been denatured under the formaldehydesystem, immobilization can take place immediately
after transfer without any pretreatment steps. The
formaldehyde has been removed from the system prior
to transfer during the gel destaining steps. If the glyoxal
denaturing system was used, the glyoxal must now be
removed from the filter. Follow one of the procedures
below to remove glyoxal from the membrane.
Option 1
1.

Air dry the membrane.

2.

Bake the membrane as follows:

Membrane Type
Nitrocellulose
Nylon

Temperature
80°C
65°C

Separation of RNA in
Agarose Gels

Returnto MainTime
2 hours under vacuum
1 - 1.5 hours

3.

Immerse the filter in 200 ml of preheated 20 mM Tris,
pH 8.0.

4.

Cool to room temperature.

Option 2
References
Ausubel, F.E., et al., Current Protocols in Molecular Biology, J. Wiley andSons, Inc.,1998.
Farrell, R.E. Jr., RNA Methodologies: A Laboratory Guide for Isolation andCharacterization, 2nd Edition, Academic Press, 1998.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, 1989.
White, H.W., et al., BioTechniques 26: 984 - 988, 1999.
Grundemann, D. & Koepsell, H., Anal. Biochem. 216: 495 - 461, 1994.

1.

Immobilize RNA on membrane either by UV irradiation or
baking.

2.

Wash membrane 65°C for 15 minutes in 20 mM Tris,
pH 8.0.

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Section VIII: Separation of RNA in Agarose Gels
FlashGel® System for RNA Analysis

The FlashGel® System completes RNA analysis in lessthan 30 minutes and requires one-fifth the amount of total
RNA for detection. The FlashGel® System is recommended
for verification and analysis of total RNA (Figure 1), quick
checks of native RNA (Figure 3) and checks for RNA
degradation (Figure 2).

1 2 3 41 2 3 41 2 3 41 2 3 4

A

B

C

D

Rapid RNA analysis
The FlashGel® System for RNA separates up to 34 samples
of RNA in 8 minutes or less. RNA samples are visible on
the FlashGel® Dock for up to 4 minutes, after which they
fade and then reappear with increasing intensity following
a 10-20 minute post-run hold. Full analysis is complete inless than 30 minutes, compared to the 1-3 hours required
for typical agarose gels. Figure 1 demonstrates analysis
time of the FlashGel® System compared to other agarose
gel methods for RNA.

Panel

Gel

A

FlashGel® System
for RNA

B

Reliant® 1.25%
MOPS Gel(Cat. #54922)

C

Reliant® 1.25%
MOPS Gel

D

Hand-cast 1%
SeaKem® LE
Agarose Gel(Cat. #50002)

Sensitive detection
RNA quantities <10 ng per band are clearly detected on
the FlashGel® System for RNA. Some RNA preparationsdeliver such low yields that there may not be the surplus
RNA needed to assess integrity. The FlashGel® System
for RNA uses a stain that reduces the amount of required
RNA by a factor of five or greater compared to ethidium
bromide. Figure 1 illustrates detection sensitivity of theFlashGel® System for RNA compared to other agarose gel
and staining methods.

Separation of RNA in
Agarose Gels

Clean, enclosed system

Sample preparation

Formaldehyde Sample
Buffer (Cat.# 50571)
Samples denatured
5 Min. at 65° C.

Run conditions

Total
time

8 min run at 225 V. Cassette
held at ambient temp for
20 min prior to imaging.

28 min60 min run at 5 V/cm. 60 min
post-stain with GelStar® Stain 120 min(Cat. #50535) 1:5000 dilution
60 min run at 5 V/cm.
60 min EtBr post-stain
and 60 min de-stain.

Denaturing gel prepared at
10µg/ml EtBr added to
2.2 M Formaldehyde in 1X
Formaldehyde Sample
Buffer. Samples denatured MOPS buffer (Cat. #50876).
60 min run at 6 V/cm.
5 Min. at 65° C.

108 min

120 minFigure1.1.Sample
Sampleof RNA
Marker(Cat. # 50575)& 2,Total
andRNA
E. coli
Total
Figureof RNA
Marker(Cat. #50575)
lanes 1 &lanes
2, and1E.coli(Ambion)Lanes
3 & 4 contain50 ng3(lanes
1 & 3 or50
250ng
ng(lanes(lanes 21&&4)3ofµl load. 2 &RNA (Ambion)Lanes& 4 containorRNA
250perng5 (lanes
4) of RNA per 5µl load.

1

2

3

4

5

6

7

The FlashGel® System for RNA eliminates the hazards
and tedium associated with RNA gel preparation. The
cassettes fully enclose the gel and running buffer,eliminating operator exposure to hazardous reagents,
and protecting samples from contaminating RNases. The
cassettes contain a 1.2% agarose and buffer blend which
are selected for purity, manufactured in a dedicated cleanroom, and guaranteed RNase-free. The FlashGel® Dock
does not come in contact with samples or gels, so it is notnecessary to dedicate a unit for RNA work.

Figure 2. Checking Sample Quality with the FlashGel® System for RNA
Sample degradation is clearly visible on the FlashGel® System for RNA.
8 min run at 225 V, followed by 20 min hold prior to imaging. Lane 1:
RNA Marker (Lonza); Lane 2: 250 ng E.coli Total RNA (Ambion); Lanes
3-7: 250 ng E.coli Total RNA incubated with increasing levels of RNase
A. Intact, denatured RNA shows sharp, clear bands on the FlashGel®
System. Partially degraded RNA has a smeared appearance, andcompletely degraded RNA appears as a low molecular weight smear.

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Section VIII: Separations of RNA in Agarose Gels
FlashGel® System for RNA Analysis — continued
1

Simple Protocol

2

3

4

5

6

7

8

Procedure
1.

Insert cassette into FlashGel® Dock.

2.

Pre-load wells with RNase-free Water(Cat. #51200).

3.

Load samples1.

4.

Plug in and turn on light and power (225 V).

5.

Run for 8 minutes2.

6.

Turn off power and hold for 10-20 minutes oruntil desired RNA bands are visible on the gel.

7.

Photograph as usual 3.
Figure 3. Quick checks of Native RNA on the FlashGel® System for RNA
Native and formaldehyde denatured RNA samples were run at 225 V
for 4 min. Native, nondenatured RNA samples do not require additional
incubation time compared to formaldehyde denatured samples, andmay be suitable for very fast checks of RNA integrity. After 20 additional
minutes, the denatured samples would absorb stain and increase invisibility.

1.

Formaldehyde Loading Buffer and RNA Markers arerecommended for best performance. Denatured RNA
samples will migrate according to their true size on
FlashGel® RNA Cassettes.

2.

The FlashGel® DNA Marker may be used to monitor
separation since it will immediately be visible on thedock in real time and be visible for the full length of therun - stop the run when the 100 bp band reaches theend of the gel. The visible blue xylene cyanol band may
also be used to track migration - stop the run when theblue band has migrated approximately 1/3 the distanceof the gel.

Lane

Sample1

RNA bands are visible for up to 4 minutes in realtime runs (see Figure 3). If desired bands are seen
within that time, then a 10-20 minute hold time prior
to imaging is not required. The longer the post-run
incubation period (up to 20 minutes), the brighter
the RNA bands become. Unlike typical agarose gelswhere samples diffuse immediately, RNA samples on
FlashGel® Cassettes remain intact for >2 hours.

3.

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Load volume

Preparation

RNA Marker (Lonza)

1.25 μl2E. coli Total RNA (Ambion)

1.25 μl3

RNA Marker

5 μl

Native samples
prepared with
FlashGel®
Loading Dye

4

E. coli Total RNA

5 μl

5

RNA Marker

1.25 μl

6E. coli Total RNA

1.25 μl7

RNA Marker

5 μl

8

E. coli Total RNA

5 μl

Formaldehyde
denaturation
at 65ºC for
5 minutes

Separation of RNA in
Agarose Gels

Returnto Main

Some components and technology of the FlashGel® System are sold underlicensing agreements. The nucleic acid stain in this product is manufactured
and sold under license from Molecular Probes, Inc., and the FlashGel®
Cassette is sold under license from Invitrogen IP Holdings, Inc, and is foruse only in research applications or quality control, and is covered bypending and issued patents. The FlashGel® Dock technology contains
Clare Chemical Research, Inc. Dark Reader® Transilluminator technology
and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250.
The electrophoresis technology is licensed from Temple University and iscovered under US Patent 6,905,585.

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