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Comparison of two different methods of processing bronchial brushings on RNA yield for transcriptomic studies in U-BIOPRED
Gibeon D1, Marriage F2, Corfield J3, Sousa A4, Sogbesan A1, Sterk PJ5, Howarth PH6, Djukanovic R6, Chung KF1, Adcock IM1, Horowitz D7, Baribaud F7
1Imperial College London – London/UK, 2The University of Manchester – Manchester/UK, 3Areteva – Nottingham/UK, 4GlaxoSmithKline – London/UK, 5University of Amsterdam Academic
Medical Center – Amsterdam/NL, 6University of Southampton – Southampton/UK, 7Janssen Research and Development – Philadelphia/US, U-BIOPRED
The Unbiased Biomarkers for the Prediction of Respiratory
Disease Outcomes (U-BIOPRED) consortium is a pan-
European public-private collaboration funded by the
Innovative Medicines Initiative (IMI) of the European Union
and EFPIA. U-BIOPRED aims to sub phenotype adult and
paediatric patients with severe refractory asthma (1) by using
an innovative systems medicine approach. The formation of
distinct handprints will enable the prediction of clinical course,
therapeutic efficacy and the identification of novel targets in
the treatment of severe asthma (2).
Transcriptomics is used to capture gene-expression profiles
of airway samples in asthma and forms an important part of
forming the U-BIOPRED handprint. However, the optimal
method for processing bronchial epithelial cells for RNA
extraction has not yet been fully established.
Background:
Methods (cont):
RNA was extracted using a modified Qiagen protocol. Samples
extracted directly into RNAlater were spun down in a 2x diluted
RNAlater solution before reverting to the standard protocol
(CIGMR-MIB-CIGMR047). RNA was quantified using a NanoDrop
1000 spectrophotometer and quality assessed using an Agilent
2100 BioAnalyser (Nano 6000 Series II Chips).
Microarray analysis: cDNA was prepared and amplified with
NuGEN Ovation V2 kits and purified using Agencourt magnetic
beads. Samples were labeled with NuGEN Encore biotin
labeling module and hybridized to Affymetrix HT HG-U133+PM
array plates and scanned with an Agilent microarray scanner.
Data was normalized by RMA and log2 transformed prior to
analysis. QC performed using Correlation and PCA analysis in
ArrayStudio (OMNIsoft corporation). Initial bioinformatics
analysis was performed using IPA® (Ingenuity® systems) .
Results:
Quality and amount of RNA
All samples had a 260/280 ratio 2.0
Concentrations ranged from 61-624ng/µl with a yield of 0.7-
18.7µg and RIN values of 6.4-8.4 (Table 1).
Electropherograms showed little sample degradation.
In Method 2, there was difficulty in pelleting the cells and
further spinning was performed after dilution with PBS at
5000 x g. In 3 samples using Method 2, bronchial brushes
were left in the tube. Comparison with samples where this
was omitted showed that while leaving the brushes in the
tube did not affect RNA quality or integrity, this resulted in a
lower RNA yield. There were no differences in the quality and
levels of RNA retrieved between the 2 Methods.
Differential gene expression
Using a general linear model–paired analysis using
ArrayStudio software (OMNIsoft corporation) we detected 131
genes upregulated between Group 2 and Group 1 (110
annotated) and 276 genes down-regulated (68 annotated)
using a ± 1.5 fold change (p value ≤ 0.05) (Table 2).
Sample
ID Conc (ng/ml)
RNA Yield
(mg) 260:280 ratio 260:230 ratio RIN
Aliquot 1 2 1 2 1 2 1 2 1 2
5930 507 393 15.2 11.8 2.08 2.12 1.89 1.61 8.4 7.8
5931 522 624 15.7 18.7 2.13 2.12 2.19 2.19 7.5 7.5
8774 78 207 2.3 6.2 2.06 2.13 2.02 1.18 7.4 6.4
8790 241 458 7.2 13.7 2.11 2.07 1.85 1.90 7.1 7.9
5975 222 284 0.7 8.5 1.92 2.09 1.41 2.15 7.1 8.0
5977 122 258 3.7 7.8 2.08 2.09 2.02 2.11 6.5 7.1
5978 61 130 1.8 3.9 1.97 2.09 1.81 1.75 7.2 7.3
Centre 1
Centre 2
Method 1= PBS wash then 1 ml RNALater
Method 2 = 5ml RNALater
Table 1 Summary of RNA extraction
Conclusions:
1. RNA samples obtained from bronchial brushes by either
method were of high quality and gave good microarray
data.
2. The process of washing the cells in PBS led to some
differences in gene expression which do not appear to
affect the downstream analysis outcomes comparing
samples from the different patient disease groups.
3. Due to the need to determine the purity of the epithelial
cells and the difficulty in pelleting cells in Method 2, we
have used Method 1 for transcriptomic studies of
bronchial epithelial cells in UBIOPRED.
Aims:
To compare RNA yield and quality achieved by two methods:
(1) Bronchoscopic samples from brushes placed in cold PBS,
spun for cell count and re-suspended in 1 ml RNAlater.
(2) Bronchoscopic samples from brushes placed directly in
suspension into 5 ml of cold RNAlater.
References:
(1) Bel EH, Sousa A, Fleming L et al. Diagnosis and definition of
severe refractory asthma: an international consensus
statement from the Innovative Medicine Initiative (IMI). Thorax
2011; 66(10):910-7.
(2) Wheelock CE, Goss VM, Balgoma D et al. Application of
‘omics technologies to biomarker discover in inflammatory lung
diseases. Eur Respir J 2013; epub ahead of print
Methods:
Seven subjects participating in the U-BIOPRED study,
including controls and asthma subjects, underwent fibreoptic
bronchoscopy at two different sites. In each patient, 4
bronchial brushings were taken from either the bronchus
intermedius and/or left main bronchus.
Two brushes were processed according to Methods 1 and 2
each. Samples were left overnight before freezing at -80oC
and then sent for RNA extraction at the central Biobank site.
Up-regulated genes Down-regulated genes
Gene Symbol Fold Change Gene Symbol Fold Change
GRP 3.54 ABCC13 -1.95
ASCL1 2.91 PRTG -1.92
SLC26A9 2.23 EPHA5 -1.77
CCL5 2.17 LECT2 -1.76
CPE 2.02 ZNF479 -1.75
FHL1 1.97 FRG1B -1.74
ID2/ID2B 1.90 FLJ35934 -1.72
JAM3 1.85 GADL1 -1.72
GPX3 1.80 LPAR4 -1.70
CD8A 1.77 GUSBP3 -1.67
Table 2. Top differentially expressed genes between
Method 2 and Method 1
Results (Cont):
RT-qPCR was performed on 109 bronchial epithelial
associated genes including those involved in IL-13, IFN and
Notch pathways, Dvorak differentiation and Woodruff
analysis. As expected only ATOH8 (1.9-fold , p value =
0.006) and FIBIN (1.4-fold , p value = .0007) were
differentially expressed between Method 2 and Method 1.
Associated Network Functions Score showed enrichment e.g. for Cellular Growth and Proliferation, Cellular Assembly and Organization, Cellular Function and Maintenance. While for Molecular and Cellular Functions, Cellular Assembly and Organization, Cellular Function and Maintenance and Cellular Movement were enriched. However, none of the enrichment was very strong and therefore does not seem to be representative of a meaningful difference.
Funded by Innovative Medicines Initiative