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Chondrocyte channel transcriptomics: do microarray data fit with expression and functional data?

Lewis R, May H, Mobasheri A, Barrett-Jolley R - Channels (Austin) (2013)

Bottom Line: We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes.The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions.We discuss whether functional or protein data exist which support the microarray data.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Biology; Institute of Ageing and Chronic Disease; Faculty of Health & Life Sciences; University of Liverpool; Liverpool, UK; The D-BOARD European Consortium for Biomarker Discovery.

ABSTRACT
To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels.

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Figure 2. Immunohistochemical identification of KCNMA1 (BK α-subunit) and KCNB1 (BK β-subunit) in sections of healthy and OA equine cartilage. The data from normal equines is reproduced with permission from Mobasheri et al.45 Macroscopically normal articular cartilage samples were obtained from weight-bearing regions of the metacarpophalangeal joints of horses of mixed breed, age, and sex. Joint tissues were sourced from an abattoir in Nantwich, Cheshire and Taunton Devon. Animals were euthanized for non-research purposes having been stunned before slaughter for meat in accordance with Welfare of Animals (Slaughter or Killing) Regulations 1995. Sections of normal (n=6) and OA (n=3) equine cartilage were probed for channel expression by immunohistochemistry essentially as previously described.45 Sections were incubated overnight at 4°C with rabbit polyclonal antibodies to the KCNMA1 and KCNB1. Antibody dilutions used ranged from 1:200 to 1:1500 in tris-buffered saline containing 1% bovine serum albumin. Slides were incubated with horseradish peroxidase-labelled polymer conjugated to affinity-purified goat anti-rabbit immunoglobulins. Cell nuclei were counterstained by incubation with aqueous haematoxylin (code no. S3309; Dako). Positive control samples were included from liver and kidney. Omission of primary antibody served as negative controls. Photomicrographs of immunostained tissue sections captured using Nikon Digital Sight DS-5M camera driven by Nikon Eclipsenet image capture software (Nikon). Positive staining is indicated by brown staining and particular evident at middle/superficial zones. (C and D) Semi-quantified protein expression density. The largest increase in expression density (from data such as that illustrated in Fig. 2) is in the middle zone, for both KCNMA1 (C) and KCNMB1 (D) (α- and β- subunit respectively). Note that in OA tissue there was insufficient superficial data to quantify expression levels.
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Figure 2: Figure 2. Immunohistochemical identification of KCNMA1 (BK α-subunit) and KCNB1 (BK β-subunit) in sections of healthy and OA equine cartilage. The data from normal equines is reproduced with permission from Mobasheri et al.45 Macroscopically normal articular cartilage samples were obtained from weight-bearing regions of the metacarpophalangeal joints of horses of mixed breed, age, and sex. Joint tissues were sourced from an abattoir in Nantwich, Cheshire and Taunton Devon. Animals were euthanized for non-research purposes having been stunned before slaughter for meat in accordance with Welfare of Animals (Slaughter or Killing) Regulations 1995. Sections of normal (n=6) and OA (n=3) equine cartilage were probed for channel expression by immunohistochemistry essentially as previously described.45 Sections were incubated overnight at 4°C with rabbit polyclonal antibodies to the KCNMA1 and KCNB1. Antibody dilutions used ranged from 1:200 to 1:1500 in tris-buffered saline containing 1% bovine serum albumin. Slides were incubated with horseradish peroxidase-labelled polymer conjugated to affinity-purified goat anti-rabbit immunoglobulins. Cell nuclei were counterstained by incubation with aqueous haematoxylin (code no. S3309; Dako). Positive control samples were included from liver and kidney. Omission of primary antibody served as negative controls. Photomicrographs of immunostained tissue sections captured using Nikon Digital Sight DS-5M camera driven by Nikon Eclipsenet image capture software (Nikon). Positive staining is indicated by brown staining and particular evident at middle/superficial zones. (C and D) Semi-quantified protein expression density. The largest increase in expression density (from data such as that illustrated in Fig. 2) is in the middle zone, for both KCNMA1 (C) and KCNMB1 (D) (α- and β- subunit respectively). Note that in OA tissue there was insufficient superficial data to quantify expression levels.

Mentions: We analysed, using immunohistochemistry, whether protein expression of BK is increased. Tissue was taken from stifle joints of horses with and without OA (see Fig. 2A and B for representative example). We investigated the expression of both α- and β-subunits of BK (only KCNMA1 was included in Karlsson 2010,50 there were no probe sets for KCNMB1 on the chip). Semi-quantitative analysis of protein expression density shows that both BK subunits were significantly increased in OA, in the middle zone (Fig. 2C and D).


Chondrocyte channel transcriptomics: do microarray data fit with expression and functional data?

Lewis R, May H, Mobasheri A, Barrett-Jolley R - Channels (Austin) (2013)

Figure 2. Immunohistochemical identification of KCNMA1 (BK α-subunit) and KCNB1 (BK β-subunit) in sections of healthy and OA equine cartilage. The data from normal equines is reproduced with permission from Mobasheri et al.45 Macroscopically normal articular cartilage samples were obtained from weight-bearing regions of the metacarpophalangeal joints of horses of mixed breed, age, and sex. Joint tissues were sourced from an abattoir in Nantwich, Cheshire and Taunton Devon. Animals were euthanized for non-research purposes having been stunned before slaughter for meat in accordance with Welfare of Animals (Slaughter or Killing) Regulations 1995. Sections of normal (n=6) and OA (n=3) equine cartilage were probed for channel expression by immunohistochemistry essentially as previously described.45 Sections were incubated overnight at 4°C with rabbit polyclonal antibodies to the KCNMA1 and KCNB1. Antibody dilutions used ranged from 1:200 to 1:1500 in tris-buffered saline containing 1% bovine serum albumin. Slides were incubated with horseradish peroxidase-labelled polymer conjugated to affinity-purified goat anti-rabbit immunoglobulins. Cell nuclei were counterstained by incubation with aqueous haematoxylin (code no. S3309; Dako). Positive control samples were included from liver and kidney. Omission of primary antibody served as negative controls. Photomicrographs of immunostained tissue sections captured using Nikon Digital Sight DS-5M camera driven by Nikon Eclipsenet image capture software (Nikon). Positive staining is indicated by brown staining and particular evident at middle/superficial zones. (C and D) Semi-quantified protein expression density. The largest increase in expression density (from data such as that illustrated in Fig. 2) is in the middle zone, for both KCNMA1 (C) and KCNMB1 (D) (α- and β- subunit respectively). Note that in OA tissue there was insufficient superficial data to quantify expression levels.
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Figure 2: Figure 2. Immunohistochemical identification of KCNMA1 (BK α-subunit) and KCNB1 (BK β-subunit) in sections of healthy and OA equine cartilage. The data from normal equines is reproduced with permission from Mobasheri et al.45 Macroscopically normal articular cartilage samples were obtained from weight-bearing regions of the metacarpophalangeal joints of horses of mixed breed, age, and sex. Joint tissues were sourced from an abattoir in Nantwich, Cheshire and Taunton Devon. Animals were euthanized for non-research purposes having been stunned before slaughter for meat in accordance with Welfare of Animals (Slaughter or Killing) Regulations 1995. Sections of normal (n=6) and OA (n=3) equine cartilage were probed for channel expression by immunohistochemistry essentially as previously described.45 Sections were incubated overnight at 4°C with rabbit polyclonal antibodies to the KCNMA1 and KCNB1. Antibody dilutions used ranged from 1:200 to 1:1500 in tris-buffered saline containing 1% bovine serum albumin. Slides were incubated with horseradish peroxidase-labelled polymer conjugated to affinity-purified goat anti-rabbit immunoglobulins. Cell nuclei were counterstained by incubation with aqueous haematoxylin (code no. S3309; Dako). Positive control samples were included from liver and kidney. Omission of primary antibody served as negative controls. Photomicrographs of immunostained tissue sections captured using Nikon Digital Sight DS-5M camera driven by Nikon Eclipsenet image capture software (Nikon). Positive staining is indicated by brown staining and particular evident at middle/superficial zones. (C and D) Semi-quantified protein expression density. The largest increase in expression density (from data such as that illustrated in Fig. 2) is in the middle zone, for both KCNMA1 (C) and KCNMB1 (D) (α- and β- subunit respectively). Note that in OA tissue there was insufficient superficial data to quantify expression levels.
Mentions: We analysed, using immunohistochemistry, whether protein expression of BK is increased. Tissue was taken from stifle joints of horses with and without OA (see Fig. 2A and B for representative example). We investigated the expression of both α- and β-subunits of BK (only KCNMA1 was included in Karlsson 2010,50 there were no probe sets for KCNMB1 on the chip). Semi-quantitative analysis of protein expression density shows that both BK subunits were significantly increased in OA, in the middle zone (Fig. 2C and D).

Bottom Line: We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes.The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions.We discuss whether functional or protein data exist which support the microarray data.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Biology; Institute of Ageing and Chronic Disease; Faculty of Health & Life Sciences; University of Liverpool; Liverpool, UK; The D-BOARD European Consortium for Biomarker Discovery.

ABSTRACT
To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels.

Show MeSH
Related in: MedlinePlus