high pure mirna isolation kit | sigma-aldrich

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High Pure miRNA Isolation Kit

y Version 06Content version: March 2013

For purification of small RNA or total RNA from animal cells, tissue, or FFPE sections

Cat. No. 05 080 576 001 Kit for 50 isolations

Store the kit at �15 to �25°C If properly stored, all kit components are

stable until the expiration date printed on the label

For life science research only. Not for use in diagnostic procedures.

www.roche-applied-science.com 2

1. What this Product Does ................................................................................................. 3Number of Tests 3Kit Contents 3Storage and Stability 3Additional Equipment and Reagents Required 4Application 4Preparation Time 4

2. How to Use this Product ................................................................................................ 52.1 Before you Begin 5

Precautions 5Handling Instructions 5Preparation of Sample Material 6Disruption and Homogenization 8Disruption and Homogenization using Rotor-Stator Homogenizers 8Disruption using a Mortar and Pestle 8Homogenization using a Syringe and Needle 8Homogenization using the MagNA Lyser Instrument 8Preparation of Working Solutions 9

2.2 Experimental Overview 102.3 Protocols for the Isolation of microRNA from Tissue 12

One-Column- Protocol (total RNA) 12Two-Column- Protocol (purified small RNA) 13

2.4 Protocol for the Isolation of microRNA from Formalin-Fixed, Paraffin-Embedded Tissue 142.5 Protocol for the Optional Treatment of the Eluate with DNase 153. Results ............................................................................................................................. 16

Purity 16Expected Yield 18

4. Troubleshooting ............................................................................................................ 195. Additional Information on this Product .................................................................... 21

How this Product Works 21Test Principle 21Product Characteristics 21References 21Quality Control 21

6. Supplementary Information ........................................................................................ 226.1 Conventions 22

Text Conventions 22Symbols 22

6.2 Changes to Previous Version 226.3 Ordering Information 236.4 Trademarks 236.5 Regulatory Disclaimer 23

What this Product Does

1. What this Product Does

Number of Tests The kit is designed for 50 purifications of small RNA (e.g., miRNA) and totalRNA containing small RNA.

Kit Contents All solutions are clear. If any solution contains a precipitate, warm thesolution prior to use at room temperature or in a 37°C water bath to dis-solve the precipitate.

Do not use vessels or pipettes containing polystyrene (PS) when workingwith the Binding Enhancer (Vial 4).

1) FFPE formalin-fixed, paraffin-embedded

Storage and Stability

Store the High Pure miRNA isolation Kit components at +15 to +25°C.If properly stored, all kit components are stable until the expiration dateprinted on the label. The kit is shipped at ambient temperature.• Improper storage of the kit at +2 to +8°C (refrigerator) or �15 to �25°C

(freezer) may lead to formation of salt precipitates in the buffers which willadversely impact the performance of the kit.

• After dissolving Proteinase K lyophilizate in Elution Buffer, aliquot and storethe solutions at �15 to �25°C. The solutions are stable for 12 months.

Vial/Cap Label Contents / Function

1white cap

Paraffin Tissue Lysis Buffer

20 ml (For FFPE1 sections only)

2pink cap

Proteinase K, Lyo 100 mg (For FFPE1 sections only)

3green cap

Binding Buffer 80 mlContains guanidine thiocyanate

4colorless cap

Binding Enhancer 20 ml

5blue cap

Wash Buffer 2 �10 ml; add 40 ml absolute ethanol to each vial Wash Buffer.

6colorless cap

Elution Buffer 30 mlWater, PCR Grade

7 High Pure Filter Tubes

2 bags with 50 columns for processing up to 700 �l sample volume.

8 Collection Tubes 2 bags with 50 polypropylene tubes (2 ml).

www.roche-applied-science.com 3High Pure miRNA Isolation Kit y Version 06

What this Product Does

Additional Equipment and Reagents Required

Refer to the list below for additional reagents and equipment required for theclean up of small RNA. • Absolute ethanol• 10 % SDS solution (For FFPE sections only)• Hemo-De or xylene (For FFPE sections only)• Standard tabletop microcentrifuge capable of 13,000 × g centrifugal force • Microcentrifuge tubes, 1.5 ml/2.0 ml, sterile• Mortar and pestle, MagNA Lyser Instrument or Rotor-Stator Homogenization

device (e.g., Ultra Turrax)• Water, PCR grade

Application The kit is designed for the isolation of small RNA (e.g., miRNA / microRNA)from animal cells, tissue samples, or formalin-fixed, paraffin-embedded tissue.It can be used to purify total RNA or to prepare samples enriched for smallRNA �100 nucleotides.The quality of the preparation is suitable for cloning, northern blotting, miRNAarray hybridization, and for the relative quantification of miRNA with RT-PCR(e.g., on the LightCycler® 480 System).

Preparation Time Total time required is approx. 30 min (without Proteinase K incubation anddeparaffinization in the preparation of paraffin-embedded tissue samples).

4 www.roche-applied-science.comHigh Pure miRNA Isolation Kit y Version 06

How to Use this Product


2. How to Use this Product

2.1 Before you Begin

Precautions Guanidine thiocyanate in Binding Buffer is an irritant. Always wear glovesand follow standard safety precautions to minimize contact when han-dling.

• Do not let these buffers touch your skin, eyes, or mucous membranes. If con-tact does occur, wash the affected area immediately with large amounts ofwater; otherwise, the reagent may cause burns. If you spill the reagent, dilutethe spill with water before wiping it up.

• Never store or use the Binding Buffer near human or animal food.• Always wear gloves and follow standard safety precautions when handling

these buffers.Guanidine thiocyanate in Binding Buffer can form toxic gases when combinedwith bleach or acid. If a spilled sample containing this solution is potentiallyinfectious, do not directly add bleach for decontamination.

Handling Instructions

RNA in sample material is subject to degradation by intracellular RNasesuntil it is fresh frozen or disrupted and homogenized in the presence ofRNase-inhibiting or denaturing agents. It is therefore imperative that• Samples are immediately flash frozen in liquid nitrogen and stored at

�60 to �80°C or are processed as soon as collected.• Frozen tissue should not be allowed to thaw during handling.• The relevant procedures should be carried out as quickly as possible.

Samples can also be stored at �60 to �80°C in 20% Binding Buffer after dis-ruption and homogenization. Yields may vary depending on storage time. Dur-ing fixation in formalin, intracellular RNases become inactivated. However,RNA is degraded and might be cross-linked to proteins. Therefore forma-lin-fixed, paraffin-embedded tissue can be stored and handled at room tem-perature. It is recommended to use sterile disposable polypropylene tubes and tips

in order to avoid RNase contamination. Wear gloves during the assay.

High Pure miRNA Isolation Kit y Version 06


Preparation of Sample Material

Samples can be derived from:• Animal tissue 1 – 50 mg either fresh deep frozen or stored in “RNAlater®”• Plant tissue 1 – 100 mg• Animal cell culture up to 106 cells• Paraffin-embedded tissue 5 – 10 �m sections• Liquid samples up to 150 �l: from cytoplasmic extracts or enzymatic reac-

tions (e.g., DNase digestion, RNA labeling, RNA ligations, in vitro transcrip-tions)

Sample materialDisruption/homogenization method of choice

Suggested procedure

Animal tissue Rotor Stator Homoge-nizer or MagNA Lyser Instrument.

• Add 400 �l 20% Binding Buffer in a sterile tube, or a MagNA Lyser Green Beads tube.

• Then add 1 – 50 mg of tissue and homogenize. Cen-trifuge lysate for 2 min at maximum speed in a micro-centrifuge and transfer the collected supernatant into a new sterile tube for Step 1 of RNA isolation.

Animal cell culturesuspension cells

Mix by pipetting up and down, homoge-nize with syringe, if necessary.

• Spin down cells at low cetrifugal force (200 × g) at +15 to +25°C for 10 min and remove supernatant.

• Then lyse cells by adding 150 �l of 20% Binding Buf-fer and homogenize by pipetting up and down until lysate appears clear.

• Pass the lysate 5 – 10 times through a 20-gauge needle (� 0.9 mm) attached to a sterile plastic syringe if the solution is not homogeneous after this step.

• Centrifuge lysate for 2 min at maximum speed in a microcentrifuge and transfer the collected superna-tant into a new sterile tube for Step 1 of RNA isola-tion.

Animal cell cultureadherent cells

Mix by pipetting up and down, homoge-nize with syringe, if necessary.

• Remove culture supernatant with a pipet. • Then lyse cells by adding 150 �l of 20% Binding Buf-

fer and homogenize by pipetting up and down until lysate appears clear.

• Pass the lysate 5 – 10 times through a 20-gauge needle (� 0.9 mm) attached to a sterile plastic syringe if the solution is not homogeneous after this step.


Alternatively, cells can be trypsinized and washed with PBS and then treated like suspension cells (see above).




Plant tissue Mortar and pestle fol-lowed by homogeniza-tion.

• Freeze the 1 – 100 mg of sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen.

• Transfer the frozen tissue powder into a sterile tube, or a MagNA Lyser Green Beads tube containing 400 �l of 20% Binding Buffer.

• Measure the amount of tissue using pre-weighed sterile caps at this step, if needed.

• Continue as quickly as possible with the homogeni-zation step using a syringe and needle or the MagNA Lyser Instrument.


Liquid samples Mix by pipetting up and down.

• Up to 150 �l of liquid can be directly used for step 1 of RNA isolation.

Formalin-fixed, par-affin-embedded tissue

Deparaffinize, then protease treatment.

• To one 5 – 10 �m section (1 cm × 1 cm) in a 1.5 ml reaction tube, add 800 �l, xylene, incubate for 5 min, and mix by overhead shaking.

• Add 400 �l ethanol abs. and mix. Centrifuge for 2 min at maximum speed and discard supernatant.

• Add 1 ml ethanol abs. and mix by overhead shaking. Centrifuge for 2 min at maximum speed and discard supernatant.

• Invert tube and blot briefly on a paper towel to get rid of residual ethanol. Dry the tissue pellet for 10 min at 55°C.

• Proceed with Step 1 of RNA isolation from formalin-fixed, paraffin-embedded tissue (2.4).

Sample materialDisruption/homogenization method of choice

Suggested procedure



Disruption and Homogenization

Efficient disruption and homogenization of the starting material is essential forintra-cellular RNA isolation procedures from tissues. The complete disruption of cell walls, extracellular material, and plasma mem-branes of cells and organelles is absolutely essential to release all the RNAcontained in the sample. Incomplete disruption results in significantly reducedyields.Homogenization is necessary to reduce the viscosity of the cell lysates pro-duced by disruption. Homogenization shears the high molecular weightgenomic DNA and other high molecular weight cellular components to createa homogeneous lysate.Incomplete homogenization results in significantly reduced yields. Some dis-ruption methods simultaneously homogenize the sample, while others requirean additional homogenization step.

Disruption and Homogenization using Rotor-Stator Homogenizers

In the presence of 20% Binding Buffer, rotor stator homogenizers thoroughlydisrupt and simultaneously homogenize tissues in 5-90 s, depending on theviscosity of the sample. By a combination of turbulence and mechanical shear-ing, the sample will be disrupted and homogenized.Keep foaming of the sample to a minimum by keeping the tip of the homoge-nizer submerged and holding the immersed tip on the side of the tube.

Disruption using a Mortar and Pestle

This step is for disruption only. Homogenization must be performed separately!For disruption using mortar and pestle, freeze the sample immediately in liquidnitrogen and grind to a fine powder under liquid nitrogen. Transfer the frozentissue powder into a tube with 20% Binding Buffer. Measure the amount of tis-sue using pre-weighed sterile caps at this step, if needed. Continue as quicklyas possible with the homogenization step using a syringe and needle or theMagNA Lyser Instrument.

Homogenization using a Syringe and Needle

After disruption, the tissue lysate can be homogenized using a syringe andneedle. High molecular weight DNA is sheared by passing the lysate througha 20-gauge needle (� 0.9 mm) attached to a sterile plastic syringe. Pass thelysate through the needle at least 5 - 10 times, or until a homogeneous lysateis achieved.

Homogenization using the MagNA Lyser Instrument

Add 400 �l 20% Binding Buffer to a MagNA Lyser Green Beads tube. Thentransfer tissue sample into the tube.Set up the MagNA Lyser Instrument as described in the Operator´s Manual.Start the disruption cycle, applying speed and time settings appropriate foryour sample material.




As an initial starting point, use the values given for some exemplary samplematerials in the table below:

#For optimal homogenization, cool samples in the MagNA Lyser RotorCooling Block (approx. 30 s, +2 to +8°C) and centrifuge for 1 min at 13,000× g to reduce foam between the disruption cycles.

Preparation of Working Solutions

In addition to the ready-to-use solutions supplied with this kit, you will need toprepare the following working solutions:

Sample Material Speed Time

Plant/Food 5,000 rpm 60 s

Liver/Kidney 6,500 rpm 50 s

Spleen/ Tumor Tissue 6,500 rpm 2 × 50 s#

Tail/ Ear/ Skin 6,500 rpm 2 – 3 × 50 s#

Content Reconstitution/ Preparation

Storage and Stability For use in

Proteinase K(Vial 2; pink cap)

Dissolve Proteinase K in 4.5 ml Elution Buffer.

Store aliquots at �15 to �25°C, stable for 12 months.

Tissue digestion for paraffin- embedded sam-ples.

Wash Buffer (Vial 5; blue cap)

Add 40 ml absolute etha-nol to Wash Buffer.Label and date bottle accordingly after adding ethanol.

Store at +15 to +25°C.Stable until expi-ration date printed on kit label.

Small RNA puri-fication: removal of large polynu-cleotides, salts, and proteins.

20% Binding Buffer

For one sample, mix 80 �l of Binding Buffer and 320 �l of nucle-ase-free, sterile, double- distilled water (or Elution Buffer) in a sterile RNase-free tube to prepare 20% Binding Buffer.For the total of 50 sam-ples, mix 4 ml of Binding Buffer and 16 ml of nucle-ase-free, sterile, dou-ble-distilled water (or Elution Buffer) in a sterile RNase-free bottle to pre-pare 20% Binding Buffer.

Store at +15 to +25°C.Stable until expi-ration date printed on kit label.

Tissue and cell disruption.




2.2 Experimental Overview

1-Column protocol for the isolation of total RNA containing small RNA

150 �l lysate supernatant Add 312 �l Binding Buffer + 200 �l Binding Enhancer

Mix well and apply mixture (max. 700 �l at a time) to a High Pure Filter Tube assem-bly, centrifuge at 13,000 × g for 30 - 60 s (repeat if lysate volume is more than 700 �l).

Discard flow through Add 500 �l Wash Buffer

Centrifuge at 13,000 × g for 30 s

Discard flow through Add 300 �l Wash Buffer

Centrifuge at 13,000 × gfor 30 s

Discard flow through

Centrifuge at 13,000 × gfor 1 min

Discard collection tube

Place the Filter Tube in a fresh 1.5 ml microcentrifuge tube

Add 50 - 100 �l Elution Buffer on the center of the filter

Incubate for 1 min at +15 to 25°C.

Centrifuge at 13,000 × g for 1 min

Purified total RNA



2-Column protocol for the isolation of small RNA �100 nucleotides

150 �l lysate supernatant

Add 312 �l Binding Buffer

Mix well and apply mixture (max. 700 �l at a time) to a High Pure Filter Tube assem-bly, centrifuge at 13,000 × g for 30 – 60 s (repeat if lysate volume is more than 700 �l).

Collect flow through

Add 200 �l Binding Enhancer

Mix well and apply mixture to a new High Pure Filter Tube

assembly, centrifuge at 13,000 × g for 30 s.


Add 500 �l Wash Buffer

Centrifuge at 13,000 × g

for 30 s


Add 300 �l Wash Buffer


for 30 s



for 1 min

Discard collection tube

Place the Filter Tube in a fresh 1.5 ml microcentrifuge

tube Add 50 - 100 �l Elution Buffer on

the center of the filter

Incubate for 1 min at +15 to 25°C.

Centrifuge at 13,000 × g for 1 min

Purified small RNA


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2.3 Protocols for the Isolation of microRNA from Tissue

One-Column- Protocol (total RNA)

� Take 150 �l of cell lysate. For optimum results, do not add lysate from more than 10 mg of

animal tissue, 50 mg of plant tissue, or 106 animal or plant cells to the column at this step.

� • Add 312 �l Binding Buffer, vortex briefly, then add 200 �l Binding Enhancer.

• Vortex 3 × 5 s.

� Combine the High Pure Filter Tube with a collection tube and pipet the whole mixture from Step 2 into the upper reservoir.

� Centrifuge for 30 s at 13,000 × g, discard the flow through. Step 3 - 4 can be repeated, in order to load the column with additional sample material (do not overload the column).

� • Add 500 �l Wash Buffer working solution.• Centrifuge for 30 s at 13,000 × g, discard the flow through.


� • Centrifuge at 13,000 × g for 1 min, in order to dry the filter fleece com-pletely.

• Place the High Pure Filter Tube into a fresh 1.5 ml microcentrifuge tube, add 100 �l Elution Buffer (Bottle 6, colorless cap), and incubate for 1 min at +15 to +25°C. To increase RNA concentration of your sample, elution with 50 �l Elu-

tion Buffer is possible. An elution step with 100 �l Elution Buffer will increase the total yield by approx. 10%.

• Centrifuge for 1 min at approx. 13,000 × g.

The microcentrifuge tube now contains the eluted total RNA. Either use 0.5 – 5 �l of the eluted RNA directly in RT-PCR or store the eluted RNA at �60 to �80°C for later analysis. Glass fibers in the eluate may interfere with optical density measure-

ment. Before determining the RNA concentration photometrically, centrifuge and transfer supernatant to a fresh 1.5 ml reaction tube.



rotocol for the Isolation of microR

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Isolation from Tissue


Two-Column- Protocol (purified small RNA)

� Take 150 �l of cell lysate. For optimum results, do not add lysate from more than 10 mg of

animal tissue, 50 mg of plant tissue, or 106 animal or plant cells to the column at this step.

� • Add 312 �l Binding Buffer. • Vortex 3 × 5 s.

� Combine the High Pure Filter Tube with a 2 ml collection tube and pipet the lysate into the upper reservoir. To minimize pipetting Steps, a 2 ml microcentrifuge tube can be used

instead of the collection tube.

� Centrifuge for 30 s at 13,000 × g in a microcentrifuge and collect the flow through.

� • Add 200 �l Binding Enhancer.• Vortex 3 × 5 s.

� Combine a new High Pure filter tube with a collection tube and pipet the whole mixture from step 5 into the upper reservoir.

� Centrifuge for 30 s at 13,000 × g, discard the flow through. Steps 3 - 7 can be repeated in order to load the column with additional sample material (do not overload the column).

• Add 500 �l Wash Buffer working solution.• Centrifuge for 30 s at 13,000 × g, discard the flow through.

• Add 300 �l Wash Buffer working solution.• Centrifuge for 30 s at 13,000 × g, discard the flow through.

� Centrifuge at 13,000 × g for 1 min, in order to dry the filter fleece com-pletely.

� • Place the High Pure Filter Tube into a fresh 1.5 ml microcentrifuge tube, add 100 �l Elution Buffer (Bottle 6, colorless cap) and incubate for 1 min at +15 to +25°C. To increase RNA concentration of your sample, elution with 50 �l

Elution Buffer is possible. An elution step with 100 �l Elution Buffer will increase the total yield by approx. 30%.

• Centrifuge for 1 min at 13,000 × g.

The microcentrifuge tube now contains the eluted microRNA. Either use 0.5 – 5 �l of the eluted RNA directly in RT-PCR or store the eluted microRNA at �60 to �80°C for later analysis.



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2.4 Protocol for the Isolation of microRNA from Formalin-Fixed, Paraffin-Embedded Tissue

It is possible to modify this protocol so it uses only 1 column; however thismay lead to higher DNA contamination. For the 1-column purification, use325 �l of Binding Enhancer in Step 6 and skip Steps 3 - 5.

� • Add 100 �l Paraffin Tissue Lysis Buffer, 16 �l 10% SDS, and 40 �l Pro-teinase K working solution to each deparaffinized sample as described above.

• Vortex 3 × 5 s.• To increase yield, incubate at 55°C for at least 3 h (up to overnight).

� • Add 325 �l Binding Buffer. • Vortex briefly.

� Add 120 �l Binding Enhancer, vortex 3 × 5 s.

� Combine the High Pure Filter Tube with a 2 ml collection tube and pipet the lysate into the upper reservoir. To minimize pipetting steps a 2 ml microcentrifuge tube can be used

instead of the collection tube.

� Centrifuge for 30 s at 13,000 × g in a microcentrifuge and collect the flow through.

� • Add 205 �l Binding Enhancer• Vortex 3 × 5 s.

� Combine a new High Pure Filter Tube with a collection tube and pipet the whole mixture from Step 6 into the upper reservoir.

Centrifuge for 30 s at 13,000 × g, discard the flow through. Steps 7 - 8 can be repeated, in order to load the column with additional sample material (do not overload the column).

• Add 500 �l Wash Buffer working solution.• Centrifuge for 30 s at 13,000 × g, discard the flowthrough.

� • Add 300 �l Wash Buffer working solution.• Centrifuge for 30 s at 13,000 × g, discard the flowthrough.

� • Centrifuge at 13,000 × g for 1 min, in order to dry the filter fleece com-pletely.

• Place the High Pure Filter Tube into a fresh 1.5 ml microcentrifuge tube, add 100 �l Elution Buffer, and incubate for 1 min at +15 to +25°C. To increase RNA concentration of your sample, elution with 50 �l Elu-

tion Buffer is possible. • Centrifuge for 1 min at 13,000 × g.

� The microcentrifuge tube now contains the eluted microRNA. Either use 0.5 – 5 �l of the eluted RNA directly in RT-PCR or store the eluted microRNA at �60 to �80°C for later analysis.



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2.5 Protocol for the Optional Treatment of the Eluate with DNase

� • For DNase treatment, it is recommended to elute the sample with 100 �l Elution Buffer to minimize loss of sample material.

• To approx. 100 �l, add 56 �l DNase Solution.• Vortex 3 × 5 s.• Incubate for 30 min at 37°C.DNase Solution: for 70 �l, mix 49.5 �l water, PCR grade, 19.5 �l 10× DNase Incubation buffer, and 1 �l DNase 1 (10 U/�l) DNase treatment on the column is not recommended as miRNA will

elute at low-salt concentrations.

� • Add 325 �l Binding Buffer. • Vortex briefly.

� Add 210 �l Binding Enhancer, vortex 3 × 5 s.

� Combine the High Pure Filter Tube with a 2 ml collection tube and pipet the DNase treated sample into the upper reservoir.

� Centrifuge for 30 s at 13,000 × g in a microcentrifuge and discard the flow through.



• Centrifuge at 13,000 × g for 1 min, in order to dry the filter fleece com-pletely.

• Place the High Pure Filter Tube into a fresh 1.5 ml microcentrifuge tube, add 100 �l Elution Buffer (Bottle 6, colorless cap) and incubate for 1 min at +15 to +25°C. To increase RNA concentration of your sample, elution with 50 �l Elu-

tion Buffer is possible. An elution step with 100 �l Elution Buffer will increase the total yield by approx. 10%.

• Centrifuge for 1 min at 13,000 × g.

� The microcentrifuge tube now contains the eluted microRNA. Either use 0.5 - 5 �l of the eluted RNA directly in RT-PCR or store the eluted microRNA at �60 to �80°C for later analysis. Glass fibers in the eluate may interfere with optical density measure-

ment. Before determining the RNA concentration photometrically, centrifuge and transfer supernatant to a fresh 1.5 ml reaction tube.


Results

3. Results

Purity • Purified small RNA (2-column protocol) is free of DNA, nucleases, and allcellular and sample contaminants that interfere with RT-PCR.

• The absence of contaminating DNA is examined by PCR without a precedingRT-reaction; no amplification product is obtained.

• The integrity and size distribution of total RNA and small RNA (2-columnprotocol) purified with the High Pure miRNA Isolation Kit have been checkedelectrophoretically on a denaturing gel (15% acrylamide/TBE/urea). A 1 �gsample of miRNA 145 was spiked into the sample (before purification).Nucleic acids were visualized by ethidium bromide staining (Fig 1).

Alternatively, electropherograms were recorded on an Agilent Bioanalyzer(Fig 2).

Fig. 1: Total RNA and purified small RNA

Eluate volume: 100 �l; 10 �l sampe per laneEluate 1: 1-Column protocol; total RNAEluate 2: 2-Column protocol; high molecular weight RNA eluted from the first filter tube(control of purification)Eluate 3: 2-Column protocol; purified small RNA (miRNA enriched)


Results


1-Column protocol; total RNA from 5 mg liver (mouse) stabilized in RNAlater®

2-Column protocol; high molecular weight RNA eluated from the first filter tube (controlof purification) from 5 mg liver (mouse) stabilized in RNAlater

2-Column protocol; purified small RNA (miRNA enriched) from 5 mg liver (mouse) stabilized in RNAlater®

Fig. 2: Total RNA and purified small RNA


Results

Expected Yield The concentration and purity of total RNA can be determined by measuringthe absorbance at 260 nm and 280 nm in a spectrophotometer. The concentration of purified small RNA: An absorbance of 1 unit at 260 nmcorresponds to 40 �g RNA per ml. The ratio between the absorbance values at260 nm and 280 nm gives an estimate of RNA purity and should be 2.0 orhigher (RNA diluted in 20 mM Tris-HCl, pH 7.5).

Type of tissue Yield of total RNA (1 column protocol) [�g/mg]

Mouse liver 1.5 - 9

Mouse kidney 0.5 - 7

Rat liver 1.5 - 8

Rat brain 0.5 - 2

Rat muscle 0.5 - 3.5

Rat heart 0.5 - 2.5

K562 cells 15 - 30 �g per 106 cells


Troubleshooting


4. Troubleshooting

Possible Cause Recommendation

Low RNA yield or purity

Kit stored under non-optimal conditions.

Store kit at +15 to +25°C at all times upon arrival.

Buffers or other reagents were exposed to conditions that reduced their effectiveness.

Store all buffers at +15 to +25°C.Close all reagent bottles tightly after each use to preserve pH, stability, and freedom from contamination.Store reconstituted Proteinase K ali-quoted at �15 to �25°C.

Ethanol not added to Wash Buf-fer.

Add absolute ethanol to the Wash Buffer before use.After adding ethanol, mix the Wash Buf-fer well and store at +15 to +25°C.Always mark Wash Buffer vial to indicate whether ethanol has been added or not.

Reagents and samples not com-pletely mixed.

Always mix the sample tube well after addition of each reagent.

Binding Enhancer not added to the lysate.

Addition of Binding Enhancer to the lysate is necessary to promote selective binding of RNA to the glass fibers.

High levels of RNase activity Be careful to create an RNase-free work-ing environment.Process sample immediately or store at �60 to �80°C until it can be processed.Use eluted RNA directly in downstream procedures or store immediately at �60 to �80°C.

Tissue homogenate is viscous and diffi-cult to pipet, low RNA yield, clogged filter tube

Insufficient disruption or homogenization.

Add more 20% Binding Buffer and repeat the homogenization step to reduce vis-cosity.

Too much sample material. Reduce the amount of sample material and/or increase the amount of 20% Binding Buffer.

DNA contamination Insufficient homogenization, too much sample material.

Perform 2-column protocol or DNase treatment.

A260 reading of product too high

Product (column eluate) contaminated with glass fibers, which scatter light.

Centrifuge the eluate for 2 min at maxi-mum speed.Transfer supernatant to a fresh 1.5 ml reaction tube without disturbing glass fibers at the bottom of the original tube.


Troubleshooting

Purified RNA sample cannot easily be loaded into the well of an agarose gel, but instead “pops out” of the well as it is loaded

Eluate containing the purified RNA product is contaminated with ethanol from the Wash Buffer.

1. After the last wash step, make certain flow through solution containing Wash Buffer does not contact the bottom of the High Pure Filter Tube.2. If this has occurred, empty the Collec-tion Tube and reinsert the contaminated filter, and recentrifuge for 30 seconds.

Incomplete elution. Elute RNA with two volumes of Elution Buffer (50 �l each): Be sure to centrifuge after each addition of Elution Buffer.

Concentration of RNA in the eluate is too low

Low amounts of RNA were added to the High Pure Filter Tube (in Step 1).

Decrease the volume of Elution Buffer used to recover RNA.Do not use less than 50 �l Elution Buffer.

Possible Cause Recommendation


Additional Information on this Product


5. Additional Information on this Product

How this Product Works

In the presence of the chaotropic salt guanidine thiocyanate, RNA binds selec-tively to special glass fibers pre-packed in the High Pure Filter Tube. BoundRNA is purified in a series of rapid wash-and-spin steps to remove contami-nating salts, proteins, and other cellular impurities, and then eluted using alow-salt solution. This simple method eliminates the need for organic solventextractions and RNA precipitation, allowing for rapid purification of manysamples simultaneously. By lowering the concentration of binding enhancer during the binding step inthe two-column protocol, the small RNA containing miRNA passes the firstcolumn unbound. When the concentration of Binding Enhancer is increased,the small RNA fraction can be bound to a second High Pure Filter Tube.

Test Principle

Product Characteristics

Recovery: The amount of RNA recovered is dependent on the amount of RNAapplied to the glass fiber fleece, the elution volume, and the nature of the tis-sue. When lysate from 1 – 10 mg mouse liver tissue or 106 K562 cells is appliedto the High Pure Filter Tube, approximately 60 – 90% of spiked miRNA can berecovered using the two-column and the one-column protocol, respectively.PCR amplification of the purified, eluted RNA in a LightCycler® 480 Instrument(miRNA16 kit from ABI) produces no signal without reverse transcription(No-RT-control).

References 1 Vogelstein, B. et al. (1979) Preparative and analytical purification of DNA from agarose Proc Natl Acad Sci USA 76 (2), 615-619.

2 Bravo V. et al. (2007) Instability of miRNA and cDNAs derivatives in RNA preparations BBRC 353, 1052-1055.

Quality Control More than 60% recovery is obtained when 1 �g miRNA is added to 106 K562cells and isolated with the one-column method, and more than 50% is isolatedwith the two-column method.

� Sample is disrupted in 20% Binding Buffer.

� RNA is isolated by binding to the glass fibers pre-packed in the High Pure Filter Tube.

� Bound RNA is washed to remove salts, proteins, and other cellular impurities.

� Purified RNA is recovered using the Elution Buffer.


Supplementary Information

6. Supplementary Information

6.1 Conventions

Text Conventions To make information consistent and memorable, the following text conven-tions are used in this document:

Symbols In this document, the following symbols are used to highlight important infor-mation:

6.2 Changes to Previous Version

• Editorial changes

Text Convention Usage

Numbered stages labeled �, �, etc.

Stages in a process that usually occur in the order listed.

Numbered instructions labeled �, �, etc.

Steps in a procedure that must be performed in the order listed.

Asterisk * Denotes a product available from Roche Applied Science.

Symbol Description

Information Note:Additional information about the current topic or procedure.

Important Note:Information critical to the success of the procedure or use of the product.


Supplementary Information


6.3 Ordering Information

Roche Applied Science offers a large selection of reagents and systems for lifescience research. For a complete overview of related products and manuals,please visit and bookmark our homepage www.roche-applied-science.comand our Special Interest Sites including: • Nucleic Acid Isolation and Purification:

http://www.roche-applied-science.com/napure

6.4 Trademarks

HIGH PURE, TRIPURE, LIGHTCYCLER, and MAGNA LYSER are trademarks ofRoche.All other product names and trademarks are the property of their respectiveowners.

6.5 Regulatory Disclaimer

For life science research only. Not for use in diagnostic procedures.

Product Pack Size Cat. No.

Associated Kits High Pure RNA Isolation Kit 50 purifications 11 828 665 001

High Pure RNA Tissue Kit 50 purifications 12 033 674 001

High Pure FFPE RNA Micro Kit 50 purifications 04 823 125 001

High Pure RNA Paraffin Kit 100 purifications 03 270 289 001

Single Reagents TriPure Isolation Reagent 50 ml200 ml

11 667 157 00111 667 165 001

DNase I rec., RNase-free 10,000 units 04 716 728 001


Contact and Support If you have questions or experience problems with this or any Roche AppliedScience (RAS) product, please contact our Technical Support staff. Ourscientists are committed to providing rapid and effective help.Please also contact us if you have suggestions for enhancing RAS productperformance or using our products in new or specialized ways. Such customerinformation has repeatedly proven invaluable to the research communityworldwide.

To ask questions, solve problems, suggest enhancements or report new appli-cations, please visit our Online Technical Support Site.

Visit the Roche Applied Science homepage, www.roche-applied-science.com, to download or request copies of the following materials:

• Instructions for Use• Material Safety Data Sheets• Certificates of Analysis• Technical Manuals• Lab FAQS: Protocols and references for life science research

To call, write, fax, or email us, visit the Roche Applied Science homepage and select your home country to display country-specific contact information.

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