Comparison of two different methods of processing ... · Comparison of two different methods of...

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Comparison of two different methods of processing bronchial brushings on RNA yield for transcriptomic studies in U-BIOPRED Gibeon D 1 , Marriage F 2 , Corfield J 3 , Sousa A 4 , Sogbesan A 1 , Sterk PJ 5 , Howarth PH 6 , Djukanovic R 6 , Chung KF 1 , Adcock IM 1 , Horowitz D 7, Baribaud F 7 1 Imperial College London London/UK, 2 The University of Manchester Manchester/UK, 3 Areteva Nottingham/UK, 4 GlaxoSmithKline London/UK, 5 University of Amsterdam Academic Medical Center Amsterdam/NL, 6 University of Southampton Southampton/UK, 7 Janssen 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 modelpaired 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 -80 o C 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

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Page 1: Comparison of two different methods of processing ... · Comparison of two different methods of processing bronchial brushings on RNA yield for transcriptomic studies in U-BIOPRED

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