Bioline ISOLATE RNA Mini Kit User manual

ISOLATE RNA Kits
Product Manual
ISOLATE RNA Mini Kit
ISOLATE Plant RNA Mini Kit

Product Manual www.bioline.com/isolate
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ISOLATE RNA Kits
TABLE OF CONTENTS
ISOLATE RNA Mini Kit 04
1. Kit contents 04
2. Description 05
3. Storage 05
4. Safety information 05
5. Product specifications 05
6. Equipment and reagents to be supplied by the user 07
7. Protocols 07
7.1 Total RNA isolation from animal tissues 07
7.2 Total RNA isolation from eukaryotic cells 09
7.3 Total RNA isolation from bacterial cells 10
8. Troubleshooting guide 12
ISOLATE Plant RNA Mini Kit 13
1. Kit contents 13
2. Description 14
3. Storage 14
4. Safety information 14
5. Product specifications 14
6. Equipment and reagents to be supplied by the user 16
7. Protocol 16
7.1 Total RNA isolation from plant tissue 16
8. Troubleshooting guide 18
General Information 19
A. Hints and tips 19
B. Technical support 21
C. Ordering information 23
D. Associated products 23
E. Product warranty and disclaimer 23

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1. KIT CONTENTS
REAGENT 10 MINIPREPS 50 MINIPREPS 250 MINIPREPS
Lysis Buffer R 6ml 30ml 125ml
Wash Buffer AR 3ml 15ml 70ml
Wash Buffer BR 2ml 8ml 40ml
RNase-free Water 1.5ml 6ml 2 x 15ml
Spin Column R1 10 50 5 x 50
Spin Column R2 10 50 5 x 50
Collection Tube 50 5 x 50 25 x 50
Elution Tube 10 50 5 x 50
Product Manual 1 1 1
Bench Protocol Sheet 1 1 1
ISOLATE RNA Mini Kit

ISOLATE RNA Kits
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RNA Mini Kit
2. DESCRIPTION
ISOLATE RNA Mini Kit is specially designed for the fast and efficient isolation of
extremely pure total RNA from a variety of samples. The kit is compatible with cultured
cells, tissues and bacterial cells. The isolated RNA is ready for downstream applications
such as reverse transcription, real-time PCR and RNA protection assays.
The protocol is easy to follow on a step-by-step basis. The cells are lysed with an
optimized Lysis Buffer, which simultaneously inactivates RNases. The lysate is then
applied to a Spin Column to selectively remove genomic DNA. There is no need to
perform a separate DNase digestion step. The lysis buffer is optimized to efficiently
lyse the cells in a chaotropic lysis buffer. The lysis buffer also inactivates RNases, thus
protecting the released RNA. The RNA is then bound to a silica membrane. Subsequent
wash steps remove the remaining cell debris. Pure RNA is eluted in the final step with
RNase-free water.
3. STORAGE
The ISOLATE RNA Mini Kit should be stored dry at room temperature. Under these
conditions, the kit is stable for 12 months.
4. SAFETY INFORMATION
Always wear gloves and a suitable lab coat when handling the reagents of this kit. For
detailed information, refer to the material data safety sheets (MSDSs) available on our
website at www.bioline.com.
5. PRODUCT SPECIFICATIONS
Starting material
• Animaltissue(upto20mg)
• Eukaryoticcells(upto5x106cells)
• Bacterialcells(upto1x109cells)
Features
• HighpurityRNA
• Rapidprotocol:15-20minutes
• Clear,easytofollowinstructions
Time required
15-20 minutes
Binding capacity
Approximately 100µg RNA
Applications
IsolationofRNAfrom:
• Animaltissue
• Eukaryoticcells
• Bacterialcells

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Lyse cells with 450µl Lysis
Buffer R
Eukaryotic cells/Bacterial cells.
Transfer supernatant to
Spin Column R1
Homogenize and lyse tissue
with 450µl Lysis Buffer R.
Transfer to 1.5ml tube.
Grind
Incubate for 3 min at RT
max. speed, 1 min
10,000 x g, 2 min
10,000 x g, 2 min
10,000 x g, 1 min
10,000 x g, 1 min
10,000 x g, 2 min
6000 x g, 1 min
Add 700µl Wash Buffer BR
Discard filtrate
Discard filtrate
Discard filtrate
Place Spin Column R2 in
Elution Tube Add 30-80µl
RNase-free water. Incubate
1 min.
Add 500µl Wash Buffer AR
Isolated RNA
Discard pellet
Discard filtrate
Discard Spin Column R1
SAVE FILTRATE
Animal tissue
Add 1 vol. 70% ethanol
to filtrate. Transfer to Spin
Column R2 in new Collection
Tube.
Total RNA isolation

ISOLATE RNA Kits
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RNA Mini Kit
6. EQUIPMENT AND REAGENTS TO BE SUPPLIED BY THE USER
• Suitablecontainertoholdsample
• MortarandpestleandliquidN2or rotor stator homogenizer
• Microcentrifugewithrotorfor1.5mland2.0mltubes
• Shakingplatform
• 70%and96-100%ethanol
• TEBuffer
• ddH2O
7. PROTOCOLS
7.1 Total RNA isolation from animal tissue
Beforeyoustart:
• Beforeusingforthersttime,add96-100%ethanoltotheWashBuffersAR
and BR as indicated on the bottles and mix.
• If any of the buffers form precipitates upon storage, re-dissolve by gently
warming. Cool to room temperature before use.
• Ifusingfrozentissue,donotallowtissuetothawduringweighingorbeforethe
addition of Lysis Buffer R. Once homogenized in the Lysis Buffer, the sample
can be stored at -20ºC for several months.
• ForinformationonhowtoworkwithRNA,readHintsandTipsonpage19.
1. Homogenize and lyse up to 20mg of tissue sample using liquid nitrogen or
a rotor-stator homogenizer.
Using liquid nitrogen
1.1. Grind the sample to a fine powder using a mortar and pestle in the
presence of liquid nitrogen. Take care that the sample does not thaw during or
after grinding.
1.2. Transfer the sample to a 1.5ml microcentrifuge tube (not supplied).
1.3. Immediately add 450µl Lysis Buffer R and homogenize the sample.
Proceed to next step. The sample can also be stored at this step at -20ºC.
Using a rotor-stator homogenizer
1.1 Transfer the sample to a suitable container.
1.2 Add 450µl Lysis Buffer R and homogenize the sample.
1.3 Transfer the sample to a 1.5ml microcentrifuge tube (not supplied).
Proceed to next step. The sample can also be stored at this step at -20ºC.

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2. Centrifuge the lysate at maximum speed for 1 minute. Place Spin Column
R1 into a 2ml Collection Tube. Carefully transfer the supernatant from
the sample to Spin Column R1. Centrifuge at 10,000 x g (12,000rpm) for 2
minutes. Discard Spin Column R1 and SAVE THE FILTRATE (for optional
DNase digestion see Hints and tips, Page 20).
Note: Ensure that there is no lysate remaining on Spin Column R1. If required, centrifuge Spin
Column R1 again until all liquid has passed through the membrane.
3. Add 1 volume (usually 400µl) of 70% ethanol to the filtrate and mix well
by pipetting. Transfer immediately to Spin Column R2 placed in a 2ml
Collection Tube. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard
the filtrate and place Spin Column R2 in a new Collection Tube.
Note: Ensure that there is no lysate remaining on Spin Column R2. If required, centrifuge Spin
Column R2 again until all liquid has passed through the membrane.
4. Add 500µl Wash Buffer AR to Spin Column R2. Centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place Spin Column R2 in
a new Collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer AR according to the instructions
on the bottle.
5. Add 700µl Wash Buffer BR to the Spin Column 2. Centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place Spin Column R2 in
a new Collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer BR according to the instructions
on the bottle.
6. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes to remove all traces of
ethanol. Discard the filtrate and place Spin Column R2 in an Elution Tube.
7. Add 30-80µl RNase-free water directly to Spin Column membrane. Incubate at
room temperature for 1 minute. Centrifuge at 6000 x g (8000rpm) for 1 minute
to elute the RNA.
Note: Use a lower volume of RNase-free water if a high concentration of RNA is required.
Increasing the volume of water will increase the yield but decrease the concentration of RNA.
Optionally, perform a second elution step to increase the yield.
8. The isolated RNA is ready for use in downstream applications or for storage
at -20ºC. For long term storage, freeze at -70ºC.

ISOLATE RNA Kits
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RNA Mini Kit
7.2 Total RNA isolation from eukaryotic cells
Beforeyoustart:
• Beforeusingforthersttime,add96-100%ethanoltotheWashBuffersAR
and BR as indicated on the bottles and mix.
• If any of the buffers form precipitates upon storage, re-dissolve by gently
warming. Cool to room temperature before use.
• Cells grown in cell culture vessels can be lysed directly in the vessel or
trypsinized to detach from the vessel. Once homogenized in the Lysis Buffer,
the sample can be stored at -20ºC for several months.
• ForinformationonhowtoworkwithRNA,readHintsandTipsonpage19.
1. Harvest up to a maximum of 5 x 106cells.
Cells grown in suspension
• Centrifugeappropriatenumberofcellsat 300xgfor5minutes.Removeall
supernatant by aspiration taking care not to disturb the pellet.
Cells grown in a monolayer
• Remove the cell culture medium completely by aspiration. Incomplete removal
of the medium will inhibit lysis of the cells and compromise the efficiency of
RNA extraction. Proceed directly to step 2.
2. Add 450µl Lysis Buffer R to the sample. Resuspend the sample completely
by pipetting up and down a few times. Incubate the sample for 3 minutes at
room temperature.
Note: No cell clumps should be visible after the lysis step to maximize RNA yield.
3. Place Spin Column R1 into a 2ml Collection Tube. Carefully transfer the
supernatant from the sample to Spin Column R1. Centrifuge at 10,000 x g
(12,000rpm) for 2 minutes. Discard Spin Column R1 and SAVE THE FILTRATE
(for optional DNase digestion see Hints and tips, Page 20).
Note: Ensure that there is no lysate remaining on Spin Column R1. If required, centrifuge the
Spin Column again until all liquid has passed through the membrane.
4. Add 1 volume of 70% ethanol to the filtrate and mix well by pipetting.
Transfer immediately to Spin Column R2 placed in a 2ml Collection Tube.
Centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard the filtrate and
place Spin Column R2 in a new Collection Tube.
Note: Ensure that there is no lysate remaining on Spin Column R2. If required, centrifuge the

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Spin Column again until all liquid has passed through the membrane.
5. Add 500µl Wash Buffer AR to Spin Column R2. Centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place Spin Column R2 in
a new Collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer AR according to the instructions
on the bottle.
6. Add 700µl Wash Buffer BR to Spin Column R2. Centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place Spin Column R2 in
a new Collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer BR according to the instructions
on the bottle.
7. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes to remove all traces of
ethanol. Discard the filtrate and place Spin Column R2 in an Elution Tube.
8. Add 30-80µl RNase-free water directly to Spin Column membrane. Incubate at
room temperature for 1 minute. Centrifuge at 6000 x g (8000rpm) for 1 minute
to elute the RNA.
Note: Use a lower volume of RNase-free water if a high concentration of RNA is required.
Increasing the volume of water will increase the yield but decrease the concentration of RNA.
Optionally, perform a second elution step to increase the yield.
9. The isolated RNA is ready for use in downstream applications or for storage
at -20ºC. For long term storage, freeze at -70ºC.
7.3 Total RNA isolation from bacterial cells
Beforeyoustart:
• Beforeusingforthersttime,add96-100%ethanoltotheWashBuffersAR
and BR as indicated on the bottles and mix.
• If any of the buffers form precipitates upon storage, re-dissolve by gently
warming. Cool to room temperature before use.
• TomaximizelysisefciencyandyieldoftotalRNA,harvestcellsinthemid-
logarithmic phase.
• Thisprotocoldescribeslysisofbacterialcellsusinglysozyme.Othermethods,
such as enzymatic lysis using Proteinase K or mechanical disruption, can also
be used.
• ForinformationonhowtoworkwithRNA,readHintsandTipsonpage19.
1. Centrifuge bacterial cells for 4 minutes at 5000 x g. Discard the supernatant
by aspiration.
Note: Up to 1 x 109cells can be processed at a time. Remove the culture medium as
much as possible without disturbing the pellet. Incomplete removal may compromise the

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RNA Mini Kit
efciency of RNA isolation.
2. Resuspend the cell pellet in 100µl TE Buffer (not supplied). Add 2-6µl
lysozyme solution (not supplied). Pipette up and down for a few times until
the solution becomes slightly viscous.
Note: For Gram –ve bacteria: Add 2µl of 20mg/ml lysozyme solution.
For Gram +ve bacteria: Add 6µl of 50mg/ml lysozyme.
3. Add 450µl Lysis Buffer R and vortex vigorously. Incubate for 3 minutes at
room temperature.
Note: To maximize total RNA yield, ensure that no cell clumps are visible.
4. Transfer the sample to Spin Column R1 placed in a 2ml Collection Tube.
Centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard Spin Column and
SAVE THE FILTRATE (for optional DNase digestion see Hints and tips, Page 20).
Note: Ensure that there is no lysate remaining on Spin Column R1. If required, centrifuge
Spin Column R1 again until all liquid has passed through the membrane.
5. Add 1 volume of 70% ethanol to the filtrate and mix well by pipetting.
6. Transfer 650µl of the sample to Spin Column R2 placed in a 2ml Collection
Tube. Centrifuge at 10,000 x g (12,000rpm) for 1 minute. Discard the filtrate
and place Spin Column R2 in a new Collection Tube. Transfer the remaining
sample from step 5 to the same Spin Column R2 and centrifuge again at
10,000 x g (12,000rpm) for 1 minute. Discard the filtrate and place Spin
Column R2 into a new Collection Tube.
Note: Ensure that there is no lysate remaining on Spin Column R2 If required, centrifuge Spin
Column R2 again until all liquid has passed through the membrane.
7. Add 500µl Wash Buffer AR and centrifuge at 10,000 x g (12,000rpm) for 1 minute.
Discard the filtrate and place Spin Column R2 into a new Collection Tube.
8. Add 700µl Wash Buffer BR and centrifuge at 10,000 x g (12,000rpm) for 1 minute.
Discard the filtrate and place Spin Column R2 into a new Collection Tube.
9. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes to remove all traces of
ethanol. Discard the filtrate and place Spin Column R2 in an Elution Tube.
10. Add 30-80µl RNase-free water directly to Spin Column membrane. Incubate at
room temperature for 1 minute. Centrifuge at 6000 x g (8000rpm) for 1 minute
to elute the RNA.
Note: Use a lower volume of RNase-free water if a high concentration of RNA is
required. Increasing the volume of water will increase the yield but decrease the
concentration of RNA. Optionally, perform a second elution step to increase the yield.
11. The isolated RNA is ready for use in downstream applications or for storage
at -20ºC. For long term storage, freeze at -70ºC.

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8. TROUBLESHOOTING GUIDE OBSERVATION POSSIBLE CAUSE RECOMMENDED VSOLUTION
Clogged Spin Column
Insufficient disruption or homogenization Reduce starting material.
Insufficient centrifugation Increase centrifugation speed and time.
Low RNA yield
Insufficient disruption or homogenization Reduce amount of starting material.
Incomplete elution Incubate sample in Elution Tube with RNase-free water
for up to 5 minutes and repeat elution step.
Low RNA concentration Highelution volume Elute RNA with a lower volume. Do not use less than
20µl.
RNA degraded
Inappropriate handling and storing of starting material Ensure proper handling and storage of samples.
Ensure that all steps are followed quickly.
RNase contamination
Ensure an RNase free working environment (see page 19). Discard any solutions contaminated with RNase during use.
DNA contamination
Incorrect lysis Check protocol has been followed correctly Too much starting material Reduce amount of starting material.
RNA does not perform well in downstream applications
Ethanol carryover during elution Increase centrifugation time for ethanol removal step Salt carryover during elution
Ensure that Wash Buffers are at room temperature. Check both solutions for salt precipitates. Resuspend any visible precipitate by gentle warming.

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ISOLATE RNA Kits
Plant RNA Mini Kit
1. KIT CONTENTS
REAGENT 10 MINIPREPS 50 MINIPREPS 250 MINIPREPS
Lysis Buffer APR 6ml 30ml 125ml
Lysis Buffer BPR 6ml 30ml 125ml
Wash Buffer APR 3ml 15ml 70ml
Wash Buffer BPR 3ml 15ml 2 x 40ml
RNase-free Water 1.5ml 6ml 2 x 15ml
Spin Column PR1 10 50 5 x 50
Spin Column PR2 10 50 5 x 50
Collection Tube 60 6 x 50 30 x 50
Elution Tube 10 50 5 x 50
Product Manual 1 1 1
Bench Protocol Sheet 1 1 1
ISOLATE Plant RNA Mini Kit

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2. DESCRIPTION
ISOLATE Plant RNA Mini Kit is specially designed for the fast and efficient isolation of
extremely pure total RNA from a variety of plant tissue samples. The isolated RNA is
ready for downstream applications such as reverse transcription, real-time PCR and
RNA protection assays.
The protocol is easy to follow on a step-by-step basis. Two lysis buffers are provided.
In most cases, Lysis Buffer APR is recommended. In cases where yield is poor with
Lysis Buffer APR, Lysis Buffer BPR should be used. Once the cells are lysed, the
lysate is applied to a Spin Column to selectively remove genomic DNA. There is no
need to perform a separate DNase digestion step. The lysis buffer is optimized to
efficiently lyse the cells by incubation in a chaotropic lysis buffer. The lysis buffer also
inactivates RNases, thus protecting the released RNA. The RNA is then bound to a
silica membrane. Subsequent wash steps remove the remaining cell debris. Pure RNA
is eluted in the final step with RNase-free water.
3. STORAGE
The ISOLATE Plant RNA Mini Kit should be stored dry at room temperature. Under
these conditions, the kit is stable for 12 months.
4. SAFETY INFORMATION
Always wear gloves and a suitable lab coat when handling the reagents of this kit. For
detailed information, refer to the material data safety sheets (MSDSs) available on our
website at www.bioline.com.
5. PRODUCT SPECIFICATIONS
Starting material
Plant tissue (up to 100mg)
Time required
30 minutes after homogenization
Features
• Rapid protocol: 30 minutes after
homogenization
• HighpurityRNA
• Clear,easytofollowinstructions
Binding capacity
Approximately 100µg RNA
Applications
IsolationofRNAfrom:
• Freshorfrozenplantmaterial

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ISOLATE RNA Kits
Plant RNA Mini Kit
10,000 x g, 2 min
10,000 x g, 1 min
10,000 x g, 1 min
Add 650µl Wash
Buffer BPR
Place Spin Column
PR2 in Elution Tube
Add 30-80µl RNase-
free water. Incubate
1 min.
Isolated plant RNA
Total RNA isolation from plant tissue
Transfer supernatant to
Spin Column PR1
Homogenize and lyse tissue
with 450µl Lysis Buffer APR
or BPR. Transfer to 1.5ml tube.
max. speed, 1 min
10,000 x g, 2 min
10,000 x g, 2 min
10,000 x g, 2 min
Add 500µl Wash Buffer
APR
Discard pellet
Discard filtrate
Discard filtrate
Discard filtrate
Discard Spin Column PR1
SAVE FILTRATE
Add 1 vol. 70% ethanol
to filtrate. Transfer to
Spin Column PR2 in new
Collection Tube.

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6. EQUIPMENT AND REAGENTS TO BE SUPPLIED BY THE USER
• Suitablecontainertoholdsample
• MortarandpestleandliquidN2or rotor stator homogenizer
• Microcentrifugewithrotorfor1.5mland2.0mltubes
• Shakingplatform
• 70%and96-100%ethanol
7. PROTOCOL
7.1 Total RNA isolation from plant tissue samples
Beforeyoustart:
• Beforeusingforthersttime,add96-100%ethanoltotheWashBuffersAR
and BR as indicated on the bottles and mix.
• Avoidfreezingandthawingofstartingmaterial.
• If any of the buffers form precipitates upon storage, re-dissolve by gently
warming. Cool to room temperature before use.
• ForinformationonhowtoworkwithRNA,readHintsandTipsonpage19.
1. Homogenize and lyse up to 100mg of fresh or frozen plant tissue sample
using liquid nitrogen or a rotor-stator homogenizer.
Using liquid nitrogen
1.1. Grind the sample to a fine powder using a mortar and pestle in the presence of
liquid nitrogen. Take care that the sample does not thaw during or after grinding.
1.2. Transfer the sample to a 1.5ml microcentrifuge tube (not supplied).
1.3. Immediately add 450µl Lysis Buffer APR or BPR and homogenize the sample.
Proceed to next step. The sample can also be stored at this step at -20ºC.
Note: Most plant material can be lysed using Lysis Buffer APR and is the recommended
buffer for all applications. In case of low RNA yield, repeat the experiment with Lysis Buffer
BPR.
Using a rotor-stator homogenizer
1.1. Transfer the sample to a suitable container.
1.2. Add 450µl Lysis Buffer APR or BPR and homogenize the sample.
Note: Most plant material can be lysed using Lysis Buffer APR and is the recommended
buffer for all applications. In case of low RNA yield, repeat the experiment with Lysis Buffer
BPR.
1.3. Transfer the sample to a 1.5ml microcentrifuge tube (not supplied). Proceed to
next step. The sample can also be stored at this step at -20ºC.

17
ISOLATE RNA Kits
Plant RNA Mini Kit
2. Centrifuge the lysate at maximum speed for 1 minute. Place Spin Column PR1
in a 2ml Collection Tube. Carefully transfer the supernatant from the sample to
Spin Column PR1. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard
Spin Column PR1 and SAVE THE FILTRATE (for optional DNase digestion see
Hints and tips, Page 20).
Note: Ensure that there is no lysate remaining on Spin Column PR1 lter. If required, centrifuge
Spin Column PR1 again until all liquid has passed through the membrane.
3. Add 1 volume (usually 400µl) of 70% ethanol to the filtrate and mix well
by pipetting. Transfer immediately to a Spin Column PR2 placed in a 2ml
Collection Tube. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard
the filtrate and place Spin Column PR2 in a new Collection Tube.
Note: Ensure that there is no lysate remaining on Spin Column PR2. If required, centrifuge Spin
Column PR2 again until all liquid has passed through the membrane.
4. Add 500µl Wash Buffer APR to Spin Column PR2. Centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place Spin Column PR2 in
a new Collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer AR according to the instructions
on the bottle.
5. Add 650µl Wash Buffer BPR to the Spin Column PR2. Centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place Spin Column PR2 in
a new Collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer BR according to the instructions
on the bottle.
6. Centrifuge at 10,000 x g (12,000rpm) for 2 minutes to remove all traces of
ethanol. Discard the filtrate and place Spin Column PR2 in an Elution Tube.
7. Add 30-80µl RNase-free water directly to the Spin Column membrane.
Incubate at room temperature for 1 minute. Centrifuge at 6000 x g (8000rpm)
for 1 minute to elute the RNA.
Note: Use a lower volume of RNase-free water if a high concentration of RNA is required.
Increasing the volume of water will increase the yield but decrease the concentration of RNA.
Optionally, perform a second elution step to increase the yield.
8. The isolated RNA is ready for use in downstream applications or for storage
at -20ºC. For long term storage, freeze at -70ºC.

Product Manual www.bioline.com/isolate
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8. TROUBLESHOOTING GUIDE OBSERVATION POSSIBLE CAUSE RECOMMENDED SOLUTION Clogged Spin Column
Insufficient disruption or homogenization Reduce starting material.
Insufficient centrifugation Increase centrifugation speed and time.
Low RNA yield
Insufficient disruption or homogenization Reduce amount of starting material.
Incomplete elution Incubate sample in Elution Tube with RNase-free water for up to 5
minutes and repeat elution step.
Low RNA concentration
Highelution volume Elute RNA with a lower volume. Do not use less than 20µl. Inappropriate handling and storing of starting material
Ensure proper handling and storage of samples. Ensure that all steps are followed quickly.
RNA degraded RNase contamination Ensure an RNase free working environment (see page 10). Discard
any solutions contaminated with RNase during use.
DNA contamination
Incorrect lysis Check protocol has been followed correctly Too much starting material Reduce amount of starting material.
RNA does not perform well in downstream applications
Ethanol carryover during elution Increase centrifugation time for ethanol removal step Salt carryover during elution
Ensure that Wash Buffers are at room temperature. Check both solutions for salt precipitates. Resuspend any visible precipitate by gentle warming.

19
ISOLATE RNA Kits
General Information
A. HINTS AND TIPS
1. Working with RNA
An RNase free environment is essential when working with RNA samples. In the
laboratory, obtaining full length, high quality RNA often proves to be a daunting task.
There are two main reasons for RNA degradation during RNA analysis. Firstly, RNA,
by its very structure, is inherently weaker than DNA. RNA is made up of ribose units,
which have a highly reactive hydroxyl group on C2 that takes part in RNA-mediated
enzymatic events. This makes RNA more chemically labile than DNA. RNA is also
more prone to heat degradation than DNA. Secondly, enzymes that degrade RNA,
ribonucleases (RNases) are so ubiquitous and hardy that getting rid of them often
proves to be nearly impossible. For example, autoclaving a solution containing bacteria
will destroy the bacterial cells, but not the RNases released from the cells.
2. Sources of RNase
• Skin: The presence of RNases on human skin surfaces has been well
documented. RNase contamination through this source is very easy to
acquire and spread if tubes, pipette tips, bench tops, etc. are touched with
bare hands.
• Dust: Dust particles oating in the air often harbor bacteria or mold. The
RNases from these microorganisms get deposited wherever the dust settles.
This includes lab equipment, open bottles, etc.
• Reagents:IfthereagentsusedforRNAanalysisarenotcertiedtobeRNase
free, there is a good chance that some of the contamination will come from
this source. Reagents can also become contaminated in the lab itself if proper
care is not taken.
• Samples:RNase contaminationcancome from thesamples themselves as
tissues and cells contain endogenous RNases.

Product Manual www.bioline.com/isolate
20
3. How to maintain an RNase-free environment
• Gloves:Alwayswearsterileglovesbeforehandlinganythingthatisgoingto
be used for RNA analysis. It is however important to remember that once the
gloves have touched equipment in the lab such as centrifuges, pipettes and
door handles, they are no longer RNase-free.
• Disposableplasticware:Disposableplasticwaregreatlyreducethepossibility
of contaminating your samples. In the event of a contamination, they also
minimize the spread of the contamination. The use of disposable tips, tubes,
etc. is therefore highly recommended.
• Good quality reagents: Always ensure that all reagents and chemical
purchased commercially are guaranteed to be RNase free. Testing each batch
before use may be a prudent step.
• DEPC-treated water: UseDEPC-treated waterinsteadof regularPCR grade
water. DEPC inactivates RNase by histidine modification of the bases. If
DEPC-treated water is made in-house, always remember to autoclave before
use to degrade the DEPC.
• RNase inhibitors: The use of RNase inhibitors is highly recommended with
samples containing endogenous RNase. Most RNase inhibitors are suitable
for use in any application where RNases are a potential problem.
• Decontamination techniques: Heat proof glassware can be baked at 180ºC
for several hours to inactive RNases. Polycarbonate or polystyrene materials
canbedecontaminatedbysoakingin3%hydrogenperoxidefor15minutes,
followed by thorough rinsing with RNase-free water.
• CorrectstorageofRNAisalsoveryimportanttoavoidRNAdegradation.In
theshortterm,RNAmaybestoredinRNase-freeH2O or TE buffer at -80ºC for
1 year without degradation. For long term storage RNA samples may be stored
asethanolprecipitatesat-20ºC.However,whendissolvedinethanol,RNAis
not dispersed evenly in the solution and cannot be used directly in quantitative
experiments. Instead, precipitates should be pelleted and redissolved in an
aqueous buffer before pipetting.