Ve r 3 .

0 HB 3520

TM
Hybrid-R miRNA

SMALL & LARGE RNA PURIFICATION HANDBOOK

Should you have any further questions, do not hesitate to contact us.
We appreciate your comments and advice.

Contact Information

Hybrid-QTM, ExprepTM, ExfectionTM, ExpinTM, ExgeneTM, GenExTM, DirExTM, Hybrid-RTM, RiboExTM,

RibospinTM, RiboclearTM, AllspinTM, RiboSaverTM, EzClearTM, EzSepTM, EzPureTM, EzPassTM, AmpONETM,
AmpMasterTM, RealAmpTM, HyperScriptTM, ProtinExTM, PAGESTATM, STEADiTM, GENTiTM, SolMateTM are
trademarks of GeneAll Biotechnology Co., Ltd.

2023 GeneAll Biotechnology, all rights reserved.

This protocol handbook is included in :

GeneAll® Hybrid-RTM miRNA (325-150)

Visit www.geneall.com for FAQ, Q&A and more information.

Brief Protocol

Homogenization

Small RNA Preparation

Phase separation

Large RNA bind

11. Add 1 vol. (usually 500 μl)
of Absolute ethanol to
pass-through

12. Apply the mixture into

Column Type W (mini)
Small RNA bind 13. Centrifuge for 30 sec,
≥10,000 x g
14. Repeat step 12~13

Small RNA wash 15. Add 500 μl Buffer RBW into mini column
16. Centrifuge for 30 sec, ≥10,000 x g
17. Add 500 μl Buffer RNW into mini column
18. Centrifuge for 30 sec, ≥10,000 x g
19. Repeat step 17~18
20. Additional centrifuge for 1 min, ≥10,000 x g
Small RNA elution

21. Apply 50 μl of Nuclease-free water into mini

column
22. Incubate for 1 min at RT
23. Centrifuge for 1 min, ≥10,000 x g
Eluate

4 GeneAll® Hybrid-RTM miRNA Protocol Handbook

1. Homogenize ~50 mg tissue samples or
1 x 107 cells in 500 μl RiboExTM
2. Incubate for 5 min at RT
3. (Optional) Centrifuge for 10 min at 4˚C,
≥12,000 x g

4. Add 100 μl chloroform

5. Incubate for 2 min at RT
6. Centrifuge for 15 min at 4˚C,
≥12,000 x g
7. Transfer the aqueous phase into a new
microcentrifuge tube
Large RNA Preparation
8. Add 1 vol. (usually 250 μl) of 50%
EtOH to transferred aqueous phase
9. Apply the mixture into Column Type B (mini)
10. Centrifuge for 30 sec, ≥10,000 x g

24. Add 500 μl Buffer SW1

into mini column
25. Centrifuge for 30 sec,
≥10,000 x g
26. Add 500 μl Buffer RNW
into mini column
27. Centrifuge for 30 sec,
Large RNA wash ≥10,000 x g
28. Repeat step 26~27
29. Additional centrifuge for
1 min, ≥10,000 x g

Large RNA elution

30. Apply 50 μl Nuclease-
free water into mini
column
31. Incubate for 1min at RT
32. Centrifuge for 1min,
Eluate ≥10,000 x g

GeneAll® Hybrid-RTM miRNA Protocol Handbook 5

6 GeneAll® Hybrid-RTM miRNA Protocol Handbook
 INDEX
Brief Protocol 04
Index 07
Kit Contents 08
Materials Not Provided
Product Specifications 09
Quality Control
Storage Conditions
Safety Information 10
Product Disclaimer
Prevention of RNase Contamination
Product Description 11
Protocol 12
Troubleshooting Guide 17
Appendix 1 19
Appendix 2 22
Appendix 3 23
Ordering Information 25

GeneAll® Hybrid-RTM miRNA Protocol Handbook 7

 Kit Contents

Cat. No. 325-150

Type mini Storage
Components Quantity
No. of preparation 50
RiboExTM 30 ml 2~8˚C
Buffer SW1 30 ml
Buffer RBW (concentrate) * 13 ml
Buffer RNW (concentrate) * † 22 ml
Nuclease-free water 15 ml Room
Column Type B (mini) (with collection tube) 50 temperature
Column Type W (mini) (with collection tube) 50 (15~25ºC)
2 ml collection tube 50
1.5 ml microcentrifuge tube 100
Protocol handbook 1
* Before first use, add absolute ethanol (ACS grade or better) into Buffer RBW and RNW as indicated on the bottle.
†
Contains sodium azide as a preservative.

Materials Not Provided

Reagents
• Absolute ethanol (ACS grade or better)
• Chloroform or 1-bromo-3-chloropropane (BCP)

Disposable materials
• RNase-free pipette tips
• Disposable gloves
• Sterile 1.5 ml microcentrifuge tubes

Equipments
• Equipment for homogenizing solid tissue
• Microcentrifuge for centrifugation at 4°C and room temperature
• Suitable protector (ex; lab coat, goggles, etc)
• Vortex mixer
8 GeneAll® Hybrid-RTM miRNA Protocol Handbook
 Product Specifications

Hybrid-RTM miRNA
Column Type B (mini) Column Type W (mini)
Specification
for Large RNA for Small RNA
Type Spin Spin

Maximum amount of starting samples Solid sample : 100 mg/prep Solid sample : 100 mg/prep
Cultured cell : 1 x 107/prep Cultured cell : 1 x 107/prep
Preparation time ≥30 min ≥30 min
Maximum loading volume of mini column 700 μl 700 μl
Minimum elution volume of mini column 50 μl 30 μl
Maximum binding capacity of mini column 100 μg 100 μg

Quality Control

All components in Hybrid-RTM miRNA are manufactured in strictly clean conditions,

and its degree of cleanness is monitored periodically. Quality control is carried out
thoroughly from lot to lot, and only the qualified kits are approved to be delivered.

Storage Conditions

All components of Hybrid-RTM miRNA (except RiboExTM solution) should be stored

at room temperature (15~25˚C). It should be protected from exposure to direct
sunlight.
RiboExTM solution should be stored at 2~8˚C for optimal performance.
During shipment or storage under cool ambient condition, a precipitate can be formed
in Buffer RBW. In such a case, heat the bottle to 50˚C to dissolve completely. Using
precipitated buffers will lead to poor DNA recovery.
Hybrid-RTM miRNA is guaranteed until the expiration date printed on the product box.

GeneAll® Hybrid-RTM miRNA Protocol Handbook 9

 Safety Information

The buffers included in Hybrid-RTM miRNA contain irritants which are harmful when in
contact with skin or eyes, or when inhaled or swallowed. Care should be taken when
handling such materials. Always wear gloves and eye protection, and follow standard
safety precautions.
RiboExTM contains phenol which is poisonous and RiboExTM, Buffer RBW, and SW1
contain chaotropic agents, which can form highly reactive compounds when combined
with bleach.
Do NOT add bleach or acidic solutions directly to the sample-preparation waste.

Product Disclaimer

Hybrid-RTM miRNA is for research use only, not for use in diagnostic procedure.

Prevention of RNase Contamination

RNase can be introduced accidentally during RNA purification. Wear disposable gloves
always, because skin often contains bacteria and molds that can be a source of RNase
contamination. Use sterile, disposable plastic wares and automatic pipettes to prevent
cross-contamination of RNase from shared equipment.

10 GeneAll® Hybrid-RTM miRNA Protocol Handbook

 Product Description

In recent years, interest in small RNA, such as siRNA and miRNA which are related
to research of gene regulation, has expanded. There are many commercial kits for
total RNA preparation, but most of these are focused on preparation of large RNA
longer than 200 nt (nucleotides). Because both siRNA and miRNA are between
15~30 nt in length, the need of specially optimized kit for small RNA (<200 nt) is
growing rapidly.
Hybrid-RTM miRNA is designed for purification of large and small RNA separately
from cultured cells or animal tissues, and co-purification in a single tube is also
available by modified protocol. This kit utilizes the lysis method of RiboExTM which
has a powerful ability of lysis and the purification method based on glass fiber
membrane technology.
Samples are homogenized in RiboExTM, a monophasic solution containing phenol
and guanidium salt, which rapidly lyse cells and inactivates nucleases. Addition
of chloroform brings about a separation of the lysate into aqueous and organic
phases. Total RNA locates in the aqueous phase while DNA and protein remain
in the interphase and organic phase. Large and small RNA in the aqueous phase is
selectively bound to Column Type B and Type W respectively. The Column Type B
selectively adsorbs the RNA larger than 200 nt in length, while the Column Type W
specifically holds the RNA smaller than 200 nt in length.
To purify large RNA, the aqueous phase is mixed with ethanol and the mixture is
applied to a Column Type B. After centrifugation, large RNA is bound to membrane
and the mixture containing small RNA goes into collection tube through the
membrane. The membrane is washed away by two wash buffers (Buffer SW1 and
Buffer RNW) and purified large RNA is eluted from the membrane by Nuclease-
free water.
To purify small RNA, the pass-through come from the binding step of large RNA
is mixed with ethanol and then applied to a Column Type W. After washing with
Buffer RBW and RNW, small RNA is eluted by Nuclease-free water.
The procedure of Hybrid-R TM miRNA takes only 30 minutes for complete
preparations of pure RNA. The purified RNA is suitable for the isolation of Poly A+
RNA, Northern blotting, dot blotting, in vitro translation, cloning, RT-PCR, RPA and
other analytical procedures.

GeneAll® Hybrid-RTM miRNA Protocol Handbook 11

 Hybrid-RTM miRNA

PROTOCOL
for large RNA and small RNA isolation

1. Homogenize ~50 mg tissue samples in 500 μl RiboExTM.

Homogenize ~1 x 107 cells in 500 μl RiboExTM.

Tissue samples
Basically, do not use more than 50 mg tissue per 0.5 ml RiboExTM
solution. Exceptionally for adipose tissue, up to 100 mg can be
used.

- Handling fresh tissue

Immediately after dissection, inactivate RNases by any one of
the following treatments.
* Homogenize in RiboExTM immediately.
* Freeze rapidly in liquid nitrogen.
* Submerge in a tissue storage buffer to protect RNA from RNases.

Cell samples
Cells grown in Monolayer RiboExTM

Pour off media, add 500 μl of RiboExTM per 10 cm2 of culture

dish area. Pass the cell lysate several times through a pipette. An
insufficient amount of RiboEx TM may result in contamination of
the isolated RNA with DNA.
Cells grown in suspension
Pellet cells by centrifugation, then lyse in 500 μl of RiboExTM per
~1 x 107 cultured cells by repetitive pipetting or vortexing.
* Do NOT wash cells before lysis with RiboExTM as this may RiboExTM
contribute to mRNA degradation.

12 GeneAll® Hybrid-RTM miRNA Protocol Handbook

2. Incubate the homogenate for 5 min at room temperature. ZZ...
ZZZZ
This step allows nucleoprotein complexes to completely
dissociate. 5 min
Homogenized samples can be stored at -70°C for at least one
month.

3. (Optional :) Centrifuge at 12,000 x g for 10 min at

4°C and transfer the supernatant to a fresh 1.5 ml
al)
tion
microcentrifuge tube (not provided). (Op
This optional step is required only for homogenate with high
contents of proteins, fats, polysaccharides or extracellular
materials, such as muscles, fat, tissue, and tuberous parts of
plants.
The resulting pellet contains extracellular membranes,
polysaccharides, and high molecular weight DNA, while the
supernatant contains RNA.
Fat tissue samples will form a layer on top of the aqueous phase.
It should be removed and discarded.
Chloroform
100 μl
4. Add 100 μl of chloroform per 500 μl of RiboExTM. Shake
vigorously for 15 sec and incubate for 2 min at room
temperature.
Alternatively, 50 μl of BCP (1-bromo-3-chloropropane) can be
used in place of chloroform.

ZZ...
5. Centrifuge at 12,000 x g for 15 min at 4°C and transfer ZZZZ

the aqueous phase to a fresh 1.5 ml microcentrifuge

2 min
tube (not provided).
The mixture will be separated into three phases; a lower phase,
an interphase, and a colorless upper aqueous phase. The upper
aqueous layer is about 50% of the volume of RiboExTM used for
homogenization.
Centrifugation at over 8°C may cause some DNA to intrude in
the aqueous phase.
15 min

GeneAll® Hybrid-RTM miRNA Protocol Handbook 13

 6. Add 1 volume (usually 250 μl) of 50% ethanol to the
transferred aqueous phase and mix thoroughly by
inverting. Do NOT centrifuge.
50% EtOH

7. Transfer all the mixture to a Column Type B (mini).

8. Centrifuge at ≥10,000 x g for 30 sec at room temperature.

Transfer the mini column to a new 2 ml collection tube
(provided), and store at room temperature. Use the pass-
through for small (micro) RNA purification. Mixture
Make sure that no mixture remains in the mini column after
centrifugation. If the residual mixture has remained, centrifuge
again at higher speed until all of the solution has pass-through.
After this step, large RNA bind to mini column and small (micro)
RNA exist in the pass-through.

Go on to step 9 for small RNA purification.

Go on to step 21 for large RNA purification.

Small (micro) RNA purification Absolute ethanol

(Blue ring column)

9. Add 1 volume (usually 500 μl) of absolute ethanol to

the collection tube including pass-through, and mix well
by pipetting. Do NOT centrifuge. Mixture

10. Transfer 650 μl of the mixture including any precipitate

to a Column Type W (mini).

11. C e n t r i f u g e a t ≥ 1 0 , 0 0 0 x g f o r 3 0 s e c a t r o o m
temperature.
Discard the pass-through and reinsert the mini column back into
the collection tube.

14 GeneAll® Hybrid-RTM miRNA Protocol Handbook

 RBW
500 μl
12. Repeat step 10~11 using the remainder of the sample.

13. Add 500 μl of Buffer RBW to the mini column.

14. C e n t r i f u g e a t ≥ 1 0 , 0 0 0 x g f o r 3 0 s e c a t r o o m
temperature.
Discard the pass-through and reinsert the mini column back into RNW
500 μl
the collection tube.

15. Add 500 μl of Buffer RNW to the mini column.

16. Centrifuge at ≥10,000 x g for 30 sec at room temperature.

Discard the pass-through and reinsert the mini column back into
the collection tube. X2

17. Repeat step 15~16.

18. Centrifuge at ≥10,000 x g for an additional 1 min at room

temperature to remove residual wash buffer. Transfer
the mini column to a new 1.5 ml microcentrifuge tube
(provided).
Residual ethanol may interfere with downstream reactions. Care
must be taken at this step for eliminating the carryover of Buffer
RNW. Nuclease-free water

19. Add 50 μl of Nuclease-free water to the center of the

membrane in the mini column. Incubate for 1 min at
room temperature.
According to the expected yield, an appropriate elution volume
can be applied on the membrane.

20. Centrifuge at ≥10,000 x g for 1 min at room temperature.

Purified small RNA can be stored at 4°C for immediate analysis
and can be stored at -70°C for long term storage.

Ready for use!

GeneAll® Hybrid-RTM miRNA Protocol Handbook 15

 Large RNA purification
(Red ring column)

SWI
21. Add 500 μl of Buffer SW1 to the Column Type B (mini).
500 μl

22. Centrifuge at ≥10,000 x g for 30 sec at room temperature.

Discard the pass-through and reinsert the mini column back into
the collection tube.

23. Add 500 μl of Buffer RNW to the mini column.

24. Centrifuge at ≥10,000 x g for 30 sec at room temperature. RNW

500 μl
Discard the pass-through and reinsert the mini column back into
the collection tube.

25. Repeat step 23~24.

26. Centrifuge at ≥10,000 x g for an additional 1 min at room

temperature to remove residual wash buffer. Transfer X2

the mini column to a new 1.5 ml microcentrifuge tube

(provided).
Residual ethanol may interfere with downstream reactions. Care
must be taken at this step for eliminating the carryover of Buffer
RNW.

27. Add 50 μl of Nuclease-free water to the center of the Nuclease-free water

membrane in the mini column. Incubate for 1 min at

room temperature.
According to the expected yield, an appropriate elution volume
can be applied on the membrane.

28. Centrifuge at ≥10,000 x g for 1 min at room temperature.

Purified large RNA can be stored at 4°C for immediate analysis and
can be stored at -70°C for long term storage.

Ready for use!

16 GeneAll® Hybrid-RTM miRNA Protocol Handbook

Troubleshooting Guide
Facts Possible Causes Suggestions
Low yield of Poor quality of Process the sample immediately after harvest
RNA starting material from animal.
Thaw the frozen sample directly in RiboExTM.

Insufficient Make sure no particulate matter remains.

homogenizing Be sure to incubate for 5 min at room
of sample temperature after homogenization.

Some aqueous phase Perform second extraction with the remaining

left aqueous phase.

Incorrect elution Add Nuclease-free water to the center of

conditions the mini column membrane.

Degradation Sample manipulated Process the sample immediately after harvest

of RNA too much before the from animal.
addition of RiboExTM
For cultured cell, minimize washing steps.
Add RiboExTM directly to plates. Do NOT
trypsinize cells.

Improper storage of Store isolated RNA at -70°C, Do NOT store

RNA at -20°C.

Reagent or disposable Make sure to use RNase-free products only.

products is not
RNase-free

Low A260/280 Aqueous phase was Avoid carryover when transferring the
(<1.6) contaminated with aqueous phase to a fresh tube.
the phenol phase

Insufficient lysis of Use 0.5 ml RiboExTM for up to 50 mg tissue

sample with or up to 1 x 107 cells.
RiboExTM

Contamination The interphase was Be sure not to transfer any of the interphase
of DNA co-transferred by (containing DNA) to the aqueous phase.
mistake

GeneAll® Hybrid-RTM miRNA Protocol Handbook 17

 Facts Possible Causes Suggestions
Contamination Insufficient RiboEx TM
Use 0.5 ml RiboExTM for 50 mg tissue or
of DNA used 1 x 107 cells.

Temperature was The phase separation should be performed at

too high during 4°C to allow optimal separating and removal
centrifugation of genomic DNA from the aqueous phase.

Cells not This can be seen After addition of RiboExTM, let cells sit 2 to
detached with some strongly 3 min. Scrape cells with a scraper. Incubate
completely adherent cells for several minutes. Collect and repeatedly
from flask pipette cells over flask surface. Then transfer
after addition homogenate to a tube.
of RiboExTM

The yield of Incorrect binding Be sure to use the proper concentrations of

miRNA is step ethanol at binding step. 50% ethanol should
too low or be used for the large RNA preparation step
miRNA do then absolute ethanol should be used for
not separate the small RNA.
completely
Too much starting Use 0.5 ml RiboExTM for 50 mg tissue or
sample 1 x 107 cells.

RNA does not Residual ethanol Centrifuge again to remove any residual
perform well remains in eluate ethanol included in Buffer RNW from mini
in downstream column membrane (step 18, 26).
application

18 GeneAll® Hybrid-RTM miRNA Protocol Handbook

 APPENDIX 1.
Co-purification of total RNA
(Large and Small RNA)

This modified protocol allows co-purification of large and small RNA.

For the purification of total RNA, separated aqueous phase is mixed with ethanol and then the
mixture is applied to Column Type W. Through this simple steps, total RNA is bound to the
membrane. After washing steps, total RNA can be eluted by Nuclease-free water.

Protocol for simultaneous purification of large RNA and small RNA

from cell samples.

1. Homogenize ~50 mg of tissue samples in 500 μl RiboExTM.

Homogenize ~1 x 107 cells in 500 μl RiboExTM.
Tissue samples
Basically, do not use more than 50 mg tissue per 0.5 ml RiboExTM solution.
But exceptionally for adipose tissue up to 100 mg can be used.

Handling fresh tissue

Immediately after dissection, inactivate RNases by any one of the following treatments.
* Homogenize in RiboExTM immediately.
* Freeze rapidly in liquid nitrogen.
* Submerge in a tissue storage buffer to protect RNA from RNases.

Cell samples
Cells grown in Monolayer
Pour off media, add 500 μl of RiboExTM per 10 cm2 of culture dish area. Pass the cell
lysate several times through a pipette. An insufficient amount of RiboExTM may result in
contamination of the isolated RNA with DNA.
Cells grown in suspension
Pellet cells by centrifugation, then lyse in 500 μl of RiboExTM per ~1 x 107 cultured cells
by repetitive pipetting or vortexing.
* Do not wash cells before lysis with RiboExTM as this may contribute to mRNA
degradation.

GeneAll® Hybrid-RTM miRNA Protocol Handbook 19

 2. Incubate the homogenate for 5 min at room temperature.
This step allows nucleoprotein complexes to completely dissociate.
Homogenized samples can be stored at -70°C for at least one month.

3. (Optional :) Centrifuge at 12,000 x g for 10 min at 4°C and transfer the

supernatant to a fresh 1.5 ml microcentrifuge tube (not provided).
This optional step is required only for homogenate with high contents of proteins, fats,
polysaccharides or extracellular materials such as muscles, fat, tissue, and tuberous
parts of plants.
The resulting pellet contains extracellular membranes, polysaccharides, and high
molecular weight DNA, while the supernatant contains RNA.
Fat tissue samples will form a layer on top of the aqueous phase.
It should be removed and discarded.

4. Add 100 μl of chloroform per 500 μl of RiboExTM. Shake vigorously for

15 sec and incubate for 2 min at room temperature.
Alternatively, 50 μl of BCP (1-bromo-3-chloropropane) can be used in place of
chloroform.

5. Centrifuge at 12,000 x g for 15 min at 4°C and transfer the aqueous

phase to a fresh 1.5 ml microcentrifuge tube (not provided).
The mixture will be separated into three phases; a lower phase, an interphase, and
a colorless upper aqueous phase. The upper aqueous phase is about 50% of the
volume of RiboExTM used for homogenization.
Centrifugation at over 8°C may cause some DNA to intrude in the aqueous phase.

6. Add 1.5 volume (usually 375 μl) of absolute ethanol to the transferred
aqueous phase and mix thoroughly by inverting. Do NOT centrifuge.

7. Transfer all the mixture including any precipitate to a Column Type W

(mini).

8. Centrifuge at ≥10,000 x g for 30 sec at room temperature.

Discard the pass-through and reinsert the mini column back into the collection tube.

9. Repeat step 7~8 using the remainder of the sample.

20 GeneAll® Hybrid-RTM miRNA Protocol Handbook

10. Add 500 μl of Buffer RBW to the mini column.

11. Centrifuge at ≥10,000 x g for 30 sec at room temperature.

Discard the pass-through and reinsert the mini column back into the collection tube.

12. Add 500 μl of Buffer RNW to the mini column.

13. Centrifuge at ≥10,000 x g for 30 sec at room temperature.

Discard the pass-through and reinsert the mini column back into the collection tube.

14. Repeat step 12~13 once more.

15. Centrifuge at ≥10,000 x g for an additional 1 min at room temperature

to remove residual wash buffer. Transfer the mini column to a new
1.5 ml microcentrifuge tube (provided).
Residual ethanol may interfere with downstream reactions. Care must be taken at this
step for eliminating the carryover of Buffer RNW.

16. Add 50 μl of Nuclease-free water to the center of the membrane in the

mini column. Incubate for 1 min at room temperature.
According to the expected yield, an appropriate elution volume can be applied on the
membrane.

17. Centrifuge at ≥10,000 x g for 1 min at room temperature.

Purified total RNA can be stored at 4°C for immediate analysis and can be stored at
-70°C for long term storage.

GeneAll® Hybrid-RTM miRNA Protocol Handbook 21

 APPENDIX 2. UV absorbance
Confirmation of RNA yield and purity by

Concentration of RNA
The concentration of RNA can be determined by the absorbance at 260 nm using spectrophotometer.
For the convenient measurement, we recommend using the NanoDrop® which can reduce
your RNA sample and time. If unavailable, you need to dilute the RNA samples to measure the
concentration through traditional spectrophotometer. The value of A260 should be between 0.15
and 1.00. Be sure to calibrate the spectrophotometer with the same solution used for dilution.
An absorbance of 1 at 260 nm is correspond to about 40 μg RNA/ml at a neutral pH. Therefore,
the concentration of RNA was calculated by the formula shown below.

A260 x dilution factor x 40=RNA μg/ml

Purity of RNA
To confirm the RNA purity, you should read the ratio of A260/A280. Pure RNA is in the range of
1.8~2.2.

22 GeneAll® Hybrid-RTM miRNA Protocol Handbook

 APPENDIX 3.
Formaldehyde agarose gel
electrophoresis (Denaturing gel method)

A denaturing agarose gel is routinely used for the assessment of the quality of an RNA preparation.
After preparation, RNA forms secondary structure via intramolecular base pairing. Therefore, it is
very difficult to get the exact result of electrophoresis because of migrating inaccuracy. However,
the denaturing gel denatures the secondary structure of RNA and makes an accurate migration.
To confirm the RNA band, the gel should be transferred to a UV transilluminator after
electrophoresis. Mainly, two RNA bands are shown. In case of animal sample, the 28S and 18S
rRNA bands are confirmed on the gel. If they are intact, the RNA bands should be sharp and the
intensity of upper band should be about twice that of the lower band.

Prepare the denaturing gel

1. Put 1 g agarose in 72 ml water and heat to dissolve thoroughly.
2. Cool to 60°C.
3. Add 10 ml of 10X MOPS buffer, 18 ml of 37% formaldehyde, and 1 μl of a 10 mg/ml
ethidium bromide (EtBr).
4. Mix well then pour the gel into the gel tray and cool to solidify it.
5. Transfer the solidified gel from tray to tank, and add enough 1X MOPS running buffer
to cover the gel.

Prepare the RNA sample

1. Make the mixture. ? μl RNA (up to 20 μg)
2 μl 10X MOPS electrophoresis buffer
4 μl formaldehyde
10 μl formamide
2. Incubate the mixture for 15 min at 65°C.
3. Chill the sample for 5 min in ice.
4. Add 2 μl of 10X formaldehyde gel-loading dye to the mixture.
5. Load the mixture in a denaturing gel which is covered with a sufficient 1X MOPS
electrophoresis buffer.
6. Run the gel and confirm the RNA band on transilluminator.
Occasionally, gel destaining may be needed to increase the visibility of the bands of RNA
in dH2O for several hours.

GeneAll® Hybrid-RTM miRNA Protocol Handbook 23

 Composition of buffers

- 10X MOPS buffer

0.2 M MOPS
20 mM sodium acetate
10 mM EDTA
pH to 7.0 with NaOH

- 10X formaldehyde gel-loading dye

50% glycerol
10 mM EDTA
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF

* Caution
When working with these chemicals, always use gloves and eye protector to avoid
contact with skin and cloth. Especially, formaldehyde and ethidium bromide (EtBr)
should be handled in a fume hood.

24 GeneAll® Hybrid-RTM miRNA Protocol Handbook

Ordering Information
Products Scale Size Cat. No. Type Products Scale Size Cat. No. Type

Hybrid-QTM for rapid preparation of plasmid DNA ExgeneTM for isolation of total DNA
50 100-150 100 105-101 spin /
Plasmid Rapidprep mini spin mini
200 100-102 250 105-152 vacuum
26 105-226 spin /
Blood SV Midi
ExprepTM for preparation of plasmid DNA 100 105-201 vacuum
50 101-150 spin / 10 105-310 spin /
mini MAXI
200 101-102 vacuum 26 105-326 vacuum
Plasmid SV 26 101-226 100 106-101 spin /
spin / mini
Midi 50 101-250 250 106-152 vacuum
vacuum Cell SV
100 101-201 10 106-310 spin /
MAXI
TM 26 106-326 vacuum
Exfection
for preparation of transfection-grade plasmid DNA 100 108-101 spin /
mini
50 111-150 spin / 250 108-152 vacuum
mini
Plasmid LE 200 111-102 vacuum 26 108-226 spin /
Clinic SV Midi
(Low Endotoxin) 26 111-226 spin / 100 108-201 vacuum
Midi
100 111-201 vacuum 10 108-310 spin /
MAXI
20 121-220 26 108-326 vacuum
Plasmid EF Midi spin
(Endotoxin Free) 100 121-201 Genomic DNA micro 50 118-050 spin
100 117-101 spin /
mini
ExpinTM for purification of fragment DNA 250 117-152 vacuum

50 102-150 spin / 26 117-226 spin /

Gel SV mini Plant SV Midi
200 102-102 vacuum 100 117-201 vacuum

50 103-150 spin / 10 117-310 spin /

PCR SV mini MAXI
200 103-102 vacuum 26 117-326 vacuum

50 113-150 Soil DNA mini mini 50 114-150 spin

spin /
CleanUp SV mini Stool DNA mini mini 50 115-150 spin
200 113-102 vacuum
Viral DNA/RNA mini 50 128-150 spin
50 112-150 spin /
Combo GP mini 50 138-150
200 112-102 vacuum FFPE Tissue DNA mini spin
250 138-152
TM
Exgene for isolation of total DNA for isolation of total DNA
GenExTM without spin column
100 104-101 spin /
mini 100 220-101
250 104-152 vacuum Sx solution
GenExTM Blood 500 220-105
26 104-226 spin /
Tissue SV Midi Lx 100 220-301 solution
100 104-201 vacuum
100 221-101
10 104-310 spin / Sx solution
MAXI GenExTM Cell 500 221-105
26 104-326 vacuum
Lx 100 221-301 solution
100 109-101 spin /
mini 100 222-101
250 109-152 vacuum Sx solution
GenExTM Tissue 500 222-105
26 109-226 spin /
Tissue Plus SV Midi Lx 100 222-301 solution
100 109-201 vacuum
10 109-310 spin /
MAXI
26 109-326 vacuum

GeneAll® Hybrid-RTM miRNA Protocol Handbook 25

 Products Scale Size Cat. No. Type Products Scale Size Cat. No. Type
for isolation of total DNA
GenExTM without spin column AmpONETM for PCR amplification
Sx 100 227-101 250 U 501-025
GenExTM Plant Mx 100 227-201 solution Taq DNA polymerase 500 U 501-050 (2.5 U/μl)
Lx 100 227-301 1,000 U 501-100
Sx 100 228-101 20 μl x 96 tubes 526-200
GenExTM Plant Plus Mx 50 228-250 solution Taq Premix solution
50 μl x 96 tubes 526-500
Lx 20 228-320

DirExTM series AmpMasterTM for PCR amplification

for preperation of PCR-template without extraction
0.5 ml x 2 tubes 541-010 solution
DirExTM 100 250-101 solution Taq Master mix
0.5 ml x 10 tubes 541-050 solution
DirExTM Fast -Tissue 96 T 260-011 solution
DirExTM Fast -Cultured cell 96 T 260-021 solution
HyperScriptTM for Reverse Transcription
DirExTM Fast -Whole blood 96 T 260-031 solution
Reverse Transcriptase 10,000 U 601-100 solution
DirExTM Fast -Blood stain 96 T 260-041 solution
TM RT Master mix 0.5 ml × 2 tubes 601-710 solution
DirEx Fast -Hair 96 T 260-051 solution
TM
DirEx Fast -Buccal swab 96 T 260-061 solution One-step RT-PCR
0.5 ml × 2 tubes 602-110 solution
Master mix
DirExTM Fast -Cigarette 96 T 260-071 solution
One-step RT-PCR
20 μl × 96 tubes 602-102 solution
Premix
RNA series for preperation of total RNA
100 301-001
RiboExTM mini solution
200 301-002 RealAmpTM for qPCR amplification
TM
Hybrid-R mini 100 305-101 spin SYBR qPCR Master 200 rxn 2 ml 801-020
solution
Hybrid-RTM Blood RNA mini 50 315-150 spin mix (2X, Low ROX) 500 rxn 5 ml 801-050
TM
Hybrid-R miRNA mini 50 325-150 spin SYBR qPCR Master 200 rxn 2 ml 801-021
solution
100 302-001 mix (2X, High ROX) 500 rxn 5 ml 801-051
RiboExTM LS mini solution
200 302-002
RiboclearTM mini 50 303-150 spin
RiboclearTM Plus mini 50 313-150 spin
RibospinTM mini 50 304-150 spin
50 314-150
RibospinTM II mini spin
300 314-103
Ribospin TM vRD mini 50 302-150 spin
Ribospin TM vRD Plus mini 50 312-150 spin
Ribospin TM vRD II mini 50 322-150 spin
TM
Ribospin Plant mini 50 307-150 spin
RibospinTM
mini 50 317-150 spin
Seed/Fruit

RibospinTM 50 314-150
mini spin
Pathogen/TNA 250 314-152
TM
Allspin mini 50 306-150 spin
RiboSaverTM mini 100 351- 001 solution

26 GeneAll® Hybrid-RTM miRNA Protocol Handbook

 Products Size Cat. No. Type Products Scale Size Cat. No. Type

Ultimately flexible
Protein series automatic extraction system

ProtinExTM Automatic extrantion equipment GTI032A system

100 ml 701-001 solution
Animal cell/tissue
48 901-048A tube
PAGESTATM Genomic DNA
Reducing 96 901-096A plate
1 ml × 10 tubes 751-001 solution
5X SDS-PAGE
48 902-048A tube
Sample Buffer Viral DNA/RNA
96 902-096A plate
for automatic nucleic acid puritication
48 903-048A tube
12 Instrument GST012 system Blood DNA
96 903-096A plate
24 Instrument GST024 system
48 904-048A tube
Genomic DNA Cell/Tissue 96 401-104 kit Plant DNA/RNA
96 904-096A plate
Genomic DNA Blood 96 402-105 kit 48 906-048A tube
LMO
Total RNA 96 404-304 kit 96 906-096A plate
Viral DNA / RNA 96 405-322 kit

CFC Seed DNA/RNA 96 406-C02 kit

Genomic DNA Plant 96 407-117 kit

Soil DNA 96 408-114 kit

Ultimately flexible
automatic extraction system

Automatic extrantion equipment GTI032 system

48 901-048 tube
Genomic DNA
96 901-096 plate

48 902-048 tube
Viral DNA/RNA
96 902-096 plate

48 903-048 tube
Whole Blood Genomic DNA
96 903-096 plate

GeneAll® Hybrid-RTM miRNA Protocol Handbook 27

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E-mail : [email protected]
Tel. 82-2-407-0096 Fax. 82-2-407-0779
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