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Analytical Protocols Summary
Introduction
The
construction of this page followed the efforts to make our results available
to the general public and investigators who are not direct participants
at GRID or the NGFN Network “Inflamation and Infection”. Information
accessible via this web site includes descriptions of standardized laboratory
and clinical procedures. Analytical protocols describe experimental procedures
such as isolating RNA and profiling gene expression in clinical samples
via microarray technology. Clinical protocols, describing standardized
procedures for collecting blood, patient data, and managing the care of
injured patients, are also available. Protocols are posted on this web
site after being validated and certified by the participating investigators.
A brief description of each numbered protocol is listed below.
Protocols on this page beginning with MMB are developed or optimised at
the Institute for Medical Microbiology.
MMB
001: Preparation of total RNA from human whole blood using the PAXgene™
Blood RNA System (PreAnalytix)
This
protocol describes the isolation of total RNA from human whole blood.
Blood volumes are drawn from venous line and collected in a room tempered
(18-25°C) PAXgene™ Blood RNA Tube, which can be stored at 4oC
or at –80oC. In this procedure, leukocytes are not separated initially.
Rather, proteins in whole blood are denatured after which nucleic acids
are precipitated directly. After lysis, total cellular RNA is separated
from DNA and proteins by precipitation and binding on a silica matrix,
according to the PAXgene™ Blood RNA Kit instructions. Isolated RNA
samples should be stored at -80°C until further analysis.
MMB
002: RNeasy® Mini Protocol for RNA Cleanup
This
protocol for RNA cleanup using the RNeasy® Mini Kit (Qiagen) is based
on the disassembly of RNA-protein aggregates and binding of RNA molecules
on a silica matrix in the presence of haotropic salts. The procedure is
meant for additional purification of RNA samples isolated via Phenol based
methods e.g. TriFast.
MMB
003: RNeasy® Mini Protocol for RNA Cleanup with on-column DNase I
digest using the RNase-Free DNase Set (Qiagen)
This
protocol describes a RNA cleanup procedure using the RNeasy® Mini
Kit (Qiagen) based on the binding of RNA molecules on a silica matrix.
The procedure implements DNase I on-column digest and is meant for additional
purification of RNA samples isolated via Phenol based methods e.g. TriFast.
MMB
004: RNeasy® Mini Protocol for Isolation of Total RNA from Animal
Cells with DNase I on-column digest
The
protocol describes the Isolation of Total RNA from Animal Cells grown
in a monolayer or suspension culture using the RNeasy® Mini Kit (Qiagen).
The Mini Kit is designed for yields of up to 100 µg of RNA using
not more then 1 x 107 cells as starting material. The cells are lysed
and homogenised in a guanidine isothiocyanate-containing buffer. The purification
is based on the disassembly of RNA-protein aggregates and binding of RNA
molecules on a silica matrix in the presence of haotropic salts. The protocol
implements DNase I on-column digest making it convenient and timesaving.
MMB
005:RNeasy Mini Protocol for Isolation of Total RNA from Animal Tissues
The
protocol describes the Isolation of Total RNA from various animal organs
and tissues. Stabilization of RNA in biological materials is necessary
because, directly after harvesting the biological sample, changes in the
gene-expression pattern occur due to specific and non-specific RNA degradation.
The RNeasy® Protect Mini Kit (Qiagen)and RNAlater RNA Stabilization
Reagent (Ambion) represent a new technology enabling rapid and reliable
preservation of gene-expression patterns in biological material so as
to provide reliable gene-expression analysis.
The Mini Kit is designed for yields of up to 100 µg of RNA using not more then 50 mg tissue as starting material. The cells are lysed and homogenised in a guanidine isothiocyanate-containing buffer. The purification is based on the disassembly of RNA-protein aggregates and binding of RNA molecules on a silica matrix in the presence of haotropic salts. The protocol implements DNase I on-column digest making it convenient and timesaving.
MMB
006: Isolation of Total RNA from Animal Tissues with peqGOLD TriFastTM
+ PLG Tubes, Eppendorf
This
is an alternative protocol for isolation of total RNA from fresh and stabilized
tissues, especially suitable for fatty tissues e.g. brain. The protocol
is based on the homogenisation and lysis of fatty tissues in a phenol
- guanidine isothiocyanate monophasic solution (PeqFGold TriFast). After
adding of chlorophorm and phase separation RNA is precipitated out of
the water phase.
MMB
007: RNA Isolation from adherent cells with peqGOLD TriFastTM + PLG Tubes,
Eppendorf
This
is an alternative protocol for stabilisation and isolation of total RNA
from Animal cells grown in a monolayer. The protocols based on the homogenisation
and lysis of fatty tissues in a phenol-guanidine isothiocyanate monophasic
solution (PeqFGold TriFast or Qiazol, Qiagen). After adding of chlorophorm
and phase separation RNA is precipitated out of the water phase.
MMB
008: Isolation of human peripheral monocytes from buffy coats using CD14
magnetic beads and a VarioMACS separator
Since
some analyses suggest variation in whole blood expression patterns depending
on the method used for cell and or RNA isolation, there is a need to identify
the contribution of individual WBC populations to gene expression patterns.
This protocol describes the isolation and enrichment of monocytes using labeling with CD14 MicroBeads and separation of positive labelled cell on a VarioMACS robot (Miltenyi Biotec). The procedure is also adaptable to manual handling and is supposed to yield a monocytic population of 98% purity.
MMB
009: In vitro infection of isolated human peripheral monocytes with Gram-
positive bacteria
A
major focus of interest for GRID (Inflammation and Infection Network of
NGFN) is to determine the effects of infection on gene expression profiles
in defined cell populations such as monocytes and microphages.
Gram-positive sepsis is becoming an increasingly prevalent threat in hospitalized patients. Bacterially produced substances, such as lipoteichoic acid, peptidoglycans, superantigen toxins, and enterotoxins, are strong inducers of inflammation in response to a Gram-positive bacterial infection. This protocol is designed to challenge donor-isolated monocytes with Gram-positive bacterial pathogens in order to monitor the cellular response.
MMB
010:Intraperitoneal infection of BALBc mice and preparation of peritoneal
macrophages for RNA isolation
Animal
models provide the possibility to study the reaction of immune system
components (cells) to the pathogen invasion in their physiological environment,
reflecting the interaction between different tissues and cell types which
is hard to mimic within in-vitro systems (monoculture). Bacterially produced
substances, such as lipoteichoic acid, peptidoglycans, superantigen toxins,
and enterotoxins, are strong inducers of inflammation in response to a
Gram-positive bacterial infection. This protocol is designed for i.p.
infection with Gram-positive bacteria and for the subsequent isolation
of mouse peritoneal macrophages.
MMB
011: Protocol for Bone Marrow-derived Macrophage Differentiation (1) fallowed
by bacterial infection or toxin treatment
A
major focus of interest for GRID (Inflammation and Infection Network of
NGFN) is to determine the effects of infection on gene expression profiles
in defined cell populations such as monocytes and microphages.
Bacterially produced substances such as endotoxins, cell wall components, superantigen toxins, and enterotoxins, are strong inducers of inflammation in response to bacterial infection.
In vitro differentiation of bone marrow cells into macrophages is an alternative to in vivo models. This protocol is designed to challenge in vitro differentiated mouse macrophages with bacterial pathogens and or bacterial toxins and antigens in order to monitor the functional response
MMB
012: Protocol for Bone Marrow-derived Macrophage Differentiation (2)
In
vitro differentiation of bone marrow cells into macrophages is an alternative
to in vivo models of infection. This protocol is designed for in vitro
differentiation of mouse bone marrow derived progenitor cells into macrophages.
MMB 013: Protocol for In vitro infection of isolated murine bone marrow derived macrophages with Shiga-Toxin producing E. coli
A
major focus of interest for GRID (Inflammation and Infection Network of
NGFN) is to determine the effects of infection on gene expression profiles
in defined cell populations such as monocytes and microphages.
Bacterially produced substances such as endotoxins, cell wall components,
superantigen toxins, and enterotoxins, are strong inducers of inflammation
in response to bacterial infection.
In vitro differentiation of bone marrow cells into macrophages is an alternative
to in vivo models. This protocol is designed to challenge in vitro differentiated
mouse macrophages with Shiga-toxin producing Escherichia coli.
MMB 014: CodeLink target labelling and array hybridisation
This procedure provides amplified and labelled cRNA molecules for hybridisation on CodeLink oligonucleotide array glass slides (Amersham biosciences).
Polyadenylated fraction of total RNA is reverse transcribed in cDNA synthesis reactions with a poly-A binding primer containing the T7-polymerase promoter. Clean up of the resulting dsDNA fragments is done using the QIAquickTM PCR Purification Kit (Qiagen, Hilden, Germany). For target labelling the cDNA is in vitro transcribed by partially substituting UTP with bio-11-UTP in the reaction mixture. Labelled cRNA is cleaned using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and 10 µg is subjected to fragmentation in the presence of Mg2+. Subsequently the fragmented targets are loaded onto glass array slides and hybridised in a shaker incubator. Washing, dying and array scan is done according to the original protocol of the manufacturer (Amersham biosciences).
MMB 015:Affymetrix Target Labeling using GeneChip® Sample Cleanup Module
This procedure provides amplified and labelled cRNA molecules for hybridisation on AffymetrixGeneChip™ for gene expression analyses.
Polyadenylated fraction of total RNA is reverse transcribed in cDNA synthesis reactions with a poly-A binding primer containing the T7-polymerase promoter. For target labelling the cDNA is in vitro transcribed by partially substituting UTP and CTP with bio-11-UTP and bio-11-CTP in the reaction mixture. Clean up of dsDNA and labelled cRNA is performed with GeneChip® Sample Cleanup Module. 15µg labelled cRNA is subjected to fragmentation and is ready for hybridisation.
MMB 016:Affymetrix Target Labeling using Qiagen purification Kits
This procedure provides amplified and labelled cRNA molecules for hybridisation on Affymetrix GeneChip™ for gene expression analyses.
Polyadenylated fraction of total RNA is reverse transcribed in cDNA synthesis reactions with a poly-A binding primer containing the T7-polymerase promoter. Clean up of the resulting dsDNA fragments is done using the QIAquickTM PCR Purification Kit (Qiagen, Hilden, Germany). For target labelling the cDNA is in vitro transcribed by partially substituting UTP and CTP with bio-11-UTP and bio-11-CTP in the reaction mixture. Labelled cRNA is cleaned using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and 15µg is subjected to fragmentation in the presence of Mg2+.
MMB 017:Affymetrix Target Labeling with limited amounts of starting material
This procedure provides amplified and labelled cRNA molecules for hybridisation on Affymetrix GeneChip™ when starting with limited amounts total RNA. For this purpose the RNA samples undergoes two rounds of amplification, which results in sufficient labelled cRNA for