Product Name : DynaMarker DIG Labeled Blue Color Marker for Small RNA(Previous name： DynaMarker Colored DIG Marker for Small RNA)
Code No. : DM270
Range : 20 - 100 bases
Size : 125 μl (5 μl × 25 loadings)
Storage : store at -20 °Cbase

Description :The DynaMarker DIG Labeled Blue Color Marker for Small
RNA consists of colored (Blue) and DIG labeled six
single-strand nucleic acids, the apparent molecularweights of which are 20, 30, 40, 50, 75 and 100 bases ofRNAs. This marker is suitable for monitoring denaturing
polyacrylamide
gelelectrophoresis
andforimmunodetection with anti-DIG antibody.
The apparent sizes of bands in DynaMarker DIG Labeled BlueColor Marker for Small RNA are in excellent agreementwith sizes of non-stained RNAs, 20, 30, 40, 50, 75 and100 bases in length (about 95 % accuracy, see Table 1).
The DynaMarker DIG Labeled Blue Color Marker for Small
RNA is supplied in a ready-to-use mixture and doesn’t
require heating or addition of a denaturing agent beforeuse.

100－75－50－40－30－20－

DynaMarker

DIG Labeled Blue Color Marker for Small RNA

Figure 1. Electrophoresis profile of DynaMarker DIG LabeledBlue Color Marker for Small RNA (5 μl) on
15 % polyacrylamide - 7.5 M urea gel / 1 ×
TBE buffer as running buffer.

Storage buffer :2 mM Tris-HCl (pH8.0), 8mM EDTA, 78 % Formamide
Quality Control :After 24-hrs incubation of the DynaMarker DIG Labeled Blue Color Marker for Small RNA at 37 °C, no visible
degradation of the marker is observed in 15 % polyacrylamide - 7.5 M urea gel electrophoresis.
Recommended loading volumes :5 - 10 μl

Electrophoresis condition :Be sure to use this marker on 10 - 15% acrylamide - Urea /1 × TBE gel and 1 × TBE as running buffer.
Under other conditions, the bands cannot be separated correctly.
Note :For accurate electrophoretic determination of molecular weights, the DynaMarker Small RNA II (code #DM192) or DynaMarker Small RNA II Easy Load (code # DM197) should be used.

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BioDynamics Laboratory Inc.
www.biodynamics.co.jp

Ver.2.3

+ 75 base RNA

DynaMarker

Small RNA IIacrylamide concentration

10 %15 %100 base

103.9 %

101.3

75*104.8

100.6

50

102.5

101.640103.0

100.5

3099.6104.5

20100.0

102.8

Table 1. This shows apparent molecular weights compared with the DynaMarker Small RNA II, and suitableacrylamide concentrations for electrophoresis of the DynaMarker DIG Labeled Blue Color Marker for Small
RNA.
(* 75 base RNA is from a newly synthesized RNA. A 75 base RNA is not included in DynaMarker Small RNA II.)

Sensitivity :This marker is suitable for chemiluminescent (e.g. CDP-star *1) immunoassay. The sensitivity depends on
the length of exposure to high speed instant film (e.g. FP-3000B *2), X-ray film or imaging instrument.
The following figure shows an example.
1212

Film: Fujifilm FP-3000B
instant black & white film
Exposure: 5 - 10 min at room temperature

Lane 1This Marker (5 μl)

Lane 2

MCF-7 total RNA (5 μg)

10 min

5 min

15 % acrylamide - 7.5 M Urea / 1 × TBE

Figure 2. Detection of DynaMarker DIG Labeled Blue Color Marker for Small RNA and hsa-miR-21.
5 μl of DynaMarker DIG Labeled Blue Color Marker for Small RNA and 5 μg of MCF-7 total RNA were
blotted onto nylon membrane, and the hsa-miR-21 was hybridized with the DIG labeled DNA probe(2.5 nM). This marker and the DIG labeled DNA probe were detected with anti-DIG-AP antibody andCDP-star.
*1: CDP-star is a trademark of Tropix, Inc.
*2: FP-3000B is a product of Fujifilm Corp.

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BioDynamics Laboratory Inc.
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Ver.2.3

Recommended usage :The DynaMarker DIG Labeled Blue Color Marker for Small RNA is suitable for monitoring denaturing
acrylamide gel electrophoresis and blotting onto membrane. One example is shown below:・Electrophoresis and blotting of DynaMarker DIG Labeled Blue Color Marker for Small RNA
1) Preparation of 12.5 % polyacrylamide - 7.5 M urea gel
40 % acrylamide : bis solution
Urea
10 × TBE
H2O

6.25 ml
9.0 g
2.0 mlto 20 ml

After urea is dissolved completely, add 20 μl of TEMED and 100 μl of 10 % ammonium persulfate. Mix
quickly then pour the gel into the mold of a vertical gel apparatus.
2) Loading and electrophoresis.
Thaw the DynaMarker DIG Labeled Blue Color Marker for Small RNA completely before use. Load thedenatured RNA sample and 5 μl of DynaMarker DIG Labeled Blue Color Marker for Small RNA into a welland run the gel using 1 × TBE electrophoresis buffer at 200 V.
3) Transfer the DynaMarker DIG Labeled Blue Color Marker for Small RNA and RNA from gel to membrane(Figure 3).
3-1) Cut a piece of positive charged nylon membrane slightly larger than the gel. Soak the membrane
and four sheets of blotting paper of appropriate size in 1 × TBE buffer.
3-2) Place two sheets of blotting paper on the anode platform of the transfer cell.
3-3) Place the membrane on top of the blotting paper.
3-4) Transfer the gel from the glass plate to the top of the membrane and press out any air bubbles.
(*Make sure that there are no air bubbles between the membrane and the gel.)3-5) Place another two sheets of blotting paper onto the gel and set the cathode assembly.
3-6) Transfer for 30 - 60 min at 2 mA/cm2.
3-7) After ensuring the marker has transferred successfully onto the membrane, remove both paper andgel. Rinse the membrane in 2 × SSC.
3-8) Fix the RNA to the membrane with a UV crosslinker.
3-9) Carry out northern hybridization (Figure 4).
References:・ Joseph Sambrook, and David W. Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.,Cold Spring Harbor Laboratory Press.
・ Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith,and Kevin Struhl (1994－) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
・ Sang Woo Kim, Zhihua Li, Patrick S. Moore, A. Paula Monaghan, Yuan Chang, Mark Nichols and BinoJohn (2010) A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids
Research. 38(7): e98

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BioDynamics Laboratory Inc.
www.biodynamics.co.jp

Ver.2.3

(1)

(2)

(3)

TransferHybridization
DIG labeled probe

Nylon membrane※The blue color bands of this marker become
pale when incubating with a hybridization

12.5 % denaturing acrylamide gelbuffer.

Figure 3. Left: Electrophoresis profile of (1) 5 μl of DynaMarker DIG Labeled Blue Color Marker for Small
RNA, (2) 5 μg of Human Breast total RNA and (3) 5 μg of Breast Adenocarcinoma(MCF-7) total RNA on 12.5 % polyacrylamide - 7.5 M urea gel / 1 × TBE buffer as
running buffer.
Center: Blotting of (1) - (3) onto nylon membrane.
Right: Hybridization with the DIG labeled DNA probes of hsa-miR-21 or hsa-miR-16 in ahybridization buffer.

Detection

Nylon membrane

miR-21

miR-16(Exposure: 10 min)Figure 4. Detection of hsa-miR-21 and hsa-miR-16
The each DIG labeled DNA probes were detected with anti-DIG-AP antibody and chemiluminescent APsubstrate.

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BioDynamics Laboratory Inc.
www.biodynamics.co.jp

Ver.2.3