Do you struggle with low yields of RNA? Do you face challenges in efficient disruption and homogenization of starting materials? This sample disruption poster provides an overview of the different disruption and homogenization methods. It provides a guide to choosing the most appropriate method for your starting material and tips for successful disruption and homogenization.

1. Friederike Krämer, Abhishek Sharma
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
Efficient disruption and homogenization: an
absolute requirement
Efficient 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.
109973411/2015
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
0
Heart 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 needle
Cell 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
Vortexing
(1
m
in)
N
o
hom
ogenization
Q
IAshredderElectric
hom
ogenizer
Starting materials Disruption Homogenization Comments
Cultured animal cells Addition of lysis buffer
Rotor–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
Yeast
Enzymatic 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.