2018/5/11 version

BioMasher I-III
Technical Document

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Advantages of BioMasher II
Extraction of Total RNA from beetle-derived antennae using BioMasherIIRNA extraction experiment using BioMasher III
Comparison of RNA extraction efficiency among BioMasher I, II, and III
Protocol for RNA extraction from mouse tissue using BioMasher I
Protocol for RNA extraction from mouse tissue using BioMasher II and III

Nippi, Incorporated
Biological and Chemical Products Division
1-1-1 Senju Midori-cho, Adachi, Tokyo 120-8601, Japan
Tel: +81-3-3888-5184, Fax: +81-3-3888-5136
The information provided in this document is intended for informational purposes only and issubject to change without notice.

Disposable homogenizer

Advantages of BioMasher II

Harvest mouse liver.
Pestle withabrasive surface
Microtube withabrasive inner surface.

Prepare the same amount of liver tissue(80 mg for each).

insufficient homogenization
Sample slips in the tube.
Sample scatters to the wall of
the tube.

Abrasive surfaces of the pestle andthe microtube allow for sufficienthomogenization in a rapid and simple
manner.

Time and labor are required forsufficient homogenization

Add buffer andhomogenize.

(Sample is degrading.)

Conventional homogenizer

BioMasher II

Centrifuge and collect sup.
(Remove fat and ppt.)

Pestle includes splashing prevention
mechanism
Sufficient homogenization iscompleted rapidly and easily.
(Suppresses degradation of sample.)

Yield of sample protein after
homogenization

1.5

Collected Amount
Increased by 20%

1.3

Splashing prevention

1.1
0.9
0.7

Abrasive surface

Conventional homogenizer

BioMasher II

Sucrose Density Gradient Ultracentrifugation(Collects rough fraction of liposome protein bysucrose density gradient ultracentrifugation)Yield of rough fraction of liposome protein
1.51.3

Collected Amount
Increased by 40%

1.1
0.90.7
Conventional homogenizer

BioMasher IIExtraction of Total RNA from beetle-derived antennae using BioMasherII

Nippi, Incorporated
Biological and Chemical Products Division[Method]
BioMasher II dramatically improves the efficiency of total RNA extraction.
Using BioMasher II, RNA was extracted from the antennae of a beetle after emergence
via the Relia Prep Nucleic Acid Purification System (Promega). The control experiment
was performed using a freeze-crush mill and 2-ml tube.Total RNA was eluted with 50 μL
of nuclease-free water from the purifying column and the absorbance was measured at 260 nm.
[Results]
The results are summarized below.

Concentration(ng/μL;50 μL)

Comparison of total RNA extracts

RNA extraction
Increased by 80%

Freeze‐Crush Mill

BioMasher II

RNA extraction experiment using BioMasher III
Nippi, Incorporated
Biological and Chemical Products Division
By BioMasher III, total RNA was extracted from liver, kidney, heart, and skeletal muscle of amouse tissues are stored in RNAlater (Ambion).
The extraction protocol is as follows.
1.

Put the tissue into the BioMasher III tube with 100 mkL of TRIzol(Thermo Fisher Scientific)
and homogenize.
2. Wash the pestle with 200 μL of TRIzol.
3. Discard the pestle and incubate 3 minutes at room temperature.
4. After centrifugation at 12,000 rpm for 30 seconds, discard the filter tube, add 700 μL ofTRIzol, and mix.
5. Add 200 μL of chloroform and incubate 3 minutes at room temperature.
6. After centrifugation at 12,000 rpm for 15 minutes at 8℃, transfer the supernatant to thenew tube.
7. Add 500 μL of isopropyl alcohol and incubate 10 minutes at room temperature.
8. After centrifugation at 12,000 rpm for 10 minutes at 8℃, discard the supernatant.
9. Add 1 mL of 75% EtOH.
10. After centrifugation at 7,500 rpm for 5 minutes at 8℃, discard the supernatant and air dryfor 5 minutes at room temperature.
11. Add 50 μL of DEPC treated water and dissolve for 10 minutes at 60℃.
12. Measure the RNA content by the absorbance at 260 nm.
Tissue1. Liver2. Kidney
3. Heart
4. Skeletal

Tissue weightmRNA

Extraction Ratio(mg)(μg/mL; 50 μL)(μg/mg)36103550

948.84931.84352.86236.52

1.32
4.66
0.50
0.24

260 nm/280 nm

1.91
1.8
1.81
1.66

MuscleExtraction was more effective with BioMasher III compared with conventional SSI pestle.
With SSI pestle, extraction ratio of liver, Kidney, Heart and Muscle are 0.92, 0.15,0.12, and 0.43,
respectively.
A agarose gel electrophoresis was performed, and the 28S and 18S bands were evaluated(RNA 1 μg/lane).

Both the 18S and 28S bands were observed in all tissues.

Comparison of RNA extraction efficiency among BioMasher I, II, and III

Nippi, Incorporated
Biological and Chemical Products Division[Method]
RNA was extracted from various organs of a mouse (Crlj:CD1 [ICR]: Adult mouse of 8 weeks orolder) using BioMasher I–III according to the attached protocol. As a control, RNA wasextracted by using a generic pestle and a 1.5 mL tube.

[Results]
The results are summarized below.
RNA Extraction Ratio (RNA (μg)/Sample (mg))
Tissue

BioMasher I

BioMasher II

BioMasher III

Generic Pestle &Liver

1.00

1.50

1.32

0.92

Kidney

0.26

1.87

4.66

0.15Cerebrum

0.16

1.14-

0.12

Spleen

0.15

5.53-

0.36

Heart

0.08

1.02

0.50

0.12

Tail

0.03

0.43-

0.16

Muscle

0.02

0.80

0.24

0.43

1.5 mL tube

RNA ug /SAMPLE mg

6.00
5.00

BioMasher I

4.00

BioMasher II

3.00
BioMasher III

2.00

Generic pestle &1.5 mL tube

1.00
0.00
liverkidneycerebrum spleenhearttailmuscle*Choose appropriate model of BioMasher for the maximum extraction ratio of RNA depending on tissue type.

Protocol for RNA Extraction from Mouse Tissue Using BioMasher I

Nippi, IncorporatedBiological and Chemical Products Division
Use a pestle with or without an O-ring of BioMasher I depending on the hardness of the tissue.
For liver, kidney, cerebrum, cerebellum, and spleen (relatively soft tissues)→ Use a pestle with an O-ring.
1
2
3
4
5

Set a filter tube into the recovery tube and put 50–100 mg of the tissue in the filter tube.
Insert the pestle with the O-ring into the filter tube and push it to the end.
Centrifuge at 15,000 x g for 30 seconds.
Discard the filter tube and pestle.
Add 1 mL of TRIzol in the recovery tube and vortex.

Follow TRIzol protocol.
6 Incubate for 5 minutes at 15ºC to 30℃.
7 Add 0.2 mL of chloroform, cap the tube, and mix by inverting by hand for 15 seconds.
8 Incubate for 2–3 minutes at 15ºC to 30℃.
9 Centrifuge at 12,000 x g for 15 minutes at 2ºC to 8℃.
10 Transfer the top RNA layer to the new tube.
11 Add 0.5 mL of isopropyl alcohol and incubate for 10 minutes at 15ºC to 30℃.
12 Centrifuge at 12,000 x g for 10 minutes at 2ºC to 8℃.
13 Discard the supernatant and add 1 mL of 75% ethanol.
14 Vortex and centrifuge at 8,000 x g for 5 minutes at 2ºC to 8℃.
15 Discard the supernatant and air dry for 5–10 minutes.
16 Add 50 μL of DEPC treated water or TE buffer and incubate for 10 minutes at 55ºC to 60℃.
For the small intestine, large intestine, lung, tail, muscle, seminal vesicle, gallbladder, salivaryglands, preputial gland, heart, and blood vessels (relatively hard tissues)→ Use a pestle without the O-ring.
123
45
6
7
89Set a filter tube into the recovery tube and put 50–100 mg of the tissue in the filter tube.
Add 500 μL of TRIzol in the filter tube and insert the pestle without the O-ring.
Disrupt the tissue by rotating the pestle while pushing it into the filter tube.
Centrifuge at 15,000 x g for 30 seconds.
Discard the pestle and add 500 μL of TRIzol to the filter tube.
Incubate for 5 minutes at 15ºC to 30℃.
Centrifuge at 15,000 x g for 30 seconds.
Discard the filter tube
Close the cap of the recovery tube and vortex.

Follow TRIzol protocol. (Nos. 6 to 16 above)Protocol for RNA extraction from mouse tissue using BioMasher II/III

Nippi, Incorporated
Biological and Chemical Products Division
BioMasher II
1 Put 50–100 mg of tissue in the tube included in BioMasher II.
2 Add 500 μL of TRIzol.
3 Insert the pestle into the tube and disrupt the tissue while pressing the pestle to theside of the tube.
4 Discard the pestle and add 500 μL of TRIzol.
5 Vortex.
Follow the TRIzol protocol.
6 Incubate for 5 minutes at 15ºC to 30℃.
7 Add 0.2 mL of chloroform, cap the tube, and mix by inverting by hand for 15 seconds.
8 Incubate for 2–3 minutes at 15ºC to 30℃.
9 Centrifuge at 12,000 x g for 15 minutes at 2ºC to 8℃.
10 Transfer the top RNA layer to the new tube.
11 Add 0.5 mL of isopropyl alcohol and incubate for 10 minutes at 15ºC to 30℃.
12 Centrifuge at 12,000 x g for 10 minutes at 2ºC to 8℃.
13 Discard the supernatant and add 1 mL of 75% ethanol.
14 Vortex and centrifuge at 8,000 x g for 5 minutes at 2ºC to 8℃.
15 Discard the supernatant and air dry for 5–10 minutes.
16 Add 50 μL of DEPC-processed water or TE buffer and incubate for 10 minutes at 55ºC
to 60℃.
BioMasher III
1 Put the sample in BioMasher III and disrupt the sample with 100 μL of TRIzol.
2 Wash out the tissue section attached to the pestle with 200 μL of TRIzol in the filter
tube.
3 Discard the pestle and allow to stand for 3 minutes at room temperature.
4 Add 700 μL of TRIzol after centrifugation at 12,000 rpm for 30 seconds at roomtemperature.
5 Vortex.
Thereafter follow the TRIzol protocol.
6 Incubate for 5 minutes at 15ºC to 30℃.
7 Add 0.2 mL of chloroform, cap the tube, and mix by inverting by hand for 15 seconds.
8 Incubate for 2–3 minutes at 15ºC to 30℃.
9 Centrifuge at 12,000 x g for 15 minutes at 2ºC to 8℃.
10 Transfer the top RNA layer to the new tube.
11 Add 0.5 mL of isopropyl alcohol and incubate for 10 minutes at 15ºC to 30℃.
12 Centrifuge at 12,000 x g for 10 minutes at 2ºC to 8℃.
13 Discard the supernatant and add 1 mL of 75% ethanol.
14 Vortex and centrifuge at 8,000 x g for 5 minutes at 2ºC to 8℃.
15 Discard the supernatant and air dry for 5–10 minutes.
16 Add 50 μL of DEPC-processed water or TE buffer and incubate for 10 minutes at 55ºC
to 60℃.