Friederike Krämer, Abhishek SharmaQIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany

Efficient disruption and homogenization: an absolute requirementEfficient disruption and homogenization of the starting material is an absolute requirement for all total RNA isolation

procedures. Disruption and homogenization are two distinct steps:

Disruption: Complete disruption of tissue structure, cell walls, and plasma membranes of cells is required to release all

the RNA contained in the sample. Different samples require different methods to achieve complete disruption. Incomplete

disruption results in significantly reduced yields.

Homogenization: Homogenization is necessary to reduce the viscosity of the cell lysates produced by disruption.

Homogenization shears the high-molecular-weight genomic DNA and other high-molecular-weight cellular components to

create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA and therefore significantly

reduced yields.

Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step.

The table on the next panel provides gives an overview of different disruption and homogenization methods suitable for

various starting materials. It can be used as a guide to choose the appropriate method for the starting material with which

you are working. The disruption and homogenization methods are described in more detail on the following panels.

Bead mills Rotor–stator homogenizers

QIAGEN solutions for disruption and homogenization

In disruption using a bead mill, the sample is agitated at high speed in the presence of beads. Disruption and simultaneous

homogenization occur by the hydrodynamic shearing and crushing action of the beads as they collide with the cells.

Disruption efficiency is influenced by:

Summary of methods

Rotor–stator homogenizers thoroughly disrupt and

simultaneously homogenize, in the presence of lysis buffer,

animal tissues in 5–90 seconds depending on the toughness

of the sample. Rotor–stator homogenizers can also be used

to homogenize cell lysates. The rotor turns at a very high

speed causing the sample to be disrupted and homogenized

by a combination of turbulence and mechanical shearing.

Foaming of the sample should be kept to a minimum by

using properly sized vessels, by keeping the tip of the

homogenizer submerged, and by holding the immersed

tip to one side of the tube. Rotor–stator homogenizers are

available in different sizes and operate with differently

sized probes. Probes with diameters of 5 mm and 7 mm

are suitable for volumes up to 300 μl and can be used for

homogenization in microfuge tubes. Probes with a diameter

of 10 mm or above require larger tubes.

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The QIAGEN guide to disruption and homogenization of starting materials for RNA isolation

Sample to Insight

• Size and composition of beads

• Ratio of buffer to beads

• Amount of starting material

The optimal beads to use are:

• 0.1 mm (mean diameter) glass beads for bacteria

• 0.5 mm glass beads for yeast and unicellular

animal cells

• 3–7 mm stainless steel beads for animal and plant

tissues

• Speed and configuration of agitator

• Disintegration time

Lung

60

50

40

30

20

10

0

TissueLyser IITissueLyser LT

BrainLiver Heart

Skin

400

300

200

100

0Heart BrainLung

TissueLyser IITissueLyser LT

DNA yields

RNA yields

B

A

For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under

liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogen-cooled, appropriately

sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue

as quickly as possible with the procedure.

Note: Grinding the sample using a mortar and pestle will disrupt the sample, but it will not homogenize it. Homogenization

must be performed separately before proceeding.

Homogenization using a syringe and needleCell and tissue lysates can be homogenized using a syringe and needle. High-molecular-weight DNA can be sheared by

passing the lysate through a 20-gauge (0.9 mm) needle, attached to a sterile plastic syringe, at least 5–10 times or until a

homogeneous lysate is achieved. Increasing the volume of lysis buffer may be required to facilitate handling and minimize

sample loss.

QIAGEN provides a range of technologies for disruption and homogenization — from QIAshredder spin columns for

fast and simple homogenization of cell lysates to TissueRuptor® and TissueLyser systems for mechanical disruption and

homogenization of tougher samples at a range of throughputs.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Trademarks: QIAGEN®, QIAcube®, DNeasy®, RNeasy®, TissueRuptor® (QIAGEN Group).

© QIAGEN 2015, all rights reserved.

It is essential that glass beads are pretreated by washing in concentrated nitric acid. Alternatively, use commercially

available acid-washed glass beads. All other disruption parameters must be determined empirically for each application.

Plant material, as well as the beads and disruption vessels, can be precooled in liquid nitrogen, and disruption should be

performed without lysis buffer. Dry, cryogenic grinding is also used for animal tissue. Cryogenic grinding (regardless of

whether in a bead mill or by mortar and pestle) does not homogenize the sample, unlike when lysis buffer is used.

QIAGEN beads for bead milling.

Mortar and pestle

Comparison of RNA yields with different homogenization methods. Northern blot of total RNA isolated from 5 x 106 HeLa cells using the RNeasy Mini procedure with the indicated homogenization methods. RNA was eluted with 2 x 40 μl water, and 10 μl was loaded per lane.

–GAPDH

Vorte

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1 min)

No hom

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QIAshred

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Electr

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izer

Starting materials Disruption Homogenization Comments

Cultured animal cells Addition of lysis bufferRotor–stator homogenizer or syringe and needle for cytoplasmic RNA

If ≤1 x 105 cells are processed lysate can be homogenized by vortexing. No homogenization protocol

Animal tissue Rotor–stator homogenizer Rotor–stator homogenizer Simultaneously disrupts and homogenizes

Animal tissue Mortar and pestle Syringe and needle Rotor–stator homogenizer and bead mills usually gives higher yields than mortar and pestle

Animal tissue Bead mill Bead mill —

Bacteria Enzymatic digestion followed by addition of lysis buffer Vortex If >5 x 108 cells are processed, further homogenization

using a syringe and needle may increase yield

Bacteria Bead mill Bead mill Bead milling simultaneously disrupts and homogenizes; bead milling cannot be replaced by vortexing

YeastEnzymatic digestion of cell wall followed by lysis of spheroplasts by lysis buffer

Vortex —

Yeast Bead mill Bead mill Bead milling simultaneously disrupts and homogenizes; bead milling cannot be replaced by vortexing

Plants and filamentous fungi Mortar and pestle Syringe and needle Mortar and pestle cannot be replaced by rotor–stator homogenizer

QIAGEN disruption and homogenization solutions.

Find out more about disruption technologies at

www.qiagen.com/Disruption

Throughput/format Product Features

Spin-column format QIAshredder For simple and rapid homogenization of:• Cell and tissue lysates

1 sample/run TissueRuptor Hand-held rotor–stator homogenizer for disruption of:• Human/animal tissues• Plant tissues

Up to 12 samples/run TissueLyser LT Compact, cost-effective bead mill for disruption of:Human/animal tissues• Plant tissues• Bacteria and yeast

Up to 48 or 192 samples/run

TissueLyser II Medium- to high-throughput bead mill for disruption of:Human/animal tissues• Plant tissues• Bacteria and yeast

Effective tissue disruption. Various rat tissues were disrupted using the TissueLyser LT or TissueLyser II. A. DNA was purified from 25 mg samples on the QIAcube® using the DNeasy® Blood and Tissue Kit. DNA yields were determined using a spectrophotometer. B. RNA was purified from 20 mg samples on the QIAcube using the RNeasy® Fibrous Tissue Mini Kit (skin, heart, and lung) or RNeasy Lipid Tissue Mini Kit (brain). RNA yields were determined using a spectrophotometer.