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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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Related in: MedlinePlus

Figure 1. Commonality of gene transcript expression between datasets. (A) Commonality between species. These represent transcripts present in each of the 3 datasets for each of the human, mouse, and rat datasets (i.e., includes 9 datasets total). Far more are observed in rodent datasets (mouse especially) than human. (B) Commonality between human derived datasets. The human studies used tissue harvested from either adolescents receiving limb length correction surgery (E-GEOD-1277), adults receiving ACL surgery (E-GEOD-16464), or post mortem (E-GEOD-10024). Samples were taken from knee (E-GEOD-16464), distal femur (E-GEOD-1277), or shoulder (E-GEOD-10024) and focussed on articular (E-GEOD-10024), mixed (E-GEOD-16464), or growth plate (E-GEOD-1277) chondrocytes. Chip Ids; E-GEOD-10024 used HG-U133A and E-GEOD-16464 used the slightly newer HG-U133A_Plus_2, but E-GEOD-1277 used the U95AV2 GeneChip. All 3 human studies used expanded chondrocytes, but E-GEOD-10024 and E-GEOD-16464 re-constituted those into 3D cultures. Extraction enzymes were collagenase P (E-GEOD-10024), clostridial collagenase and deoxyribonuclease I (E-GEOD-16464) and trypsin (E-GEOD-1277). (C) Commonality between mouse derived datasets. Rodent studies suffer from inherent difficulties in extraction of tissue since cartilage is thinner than larger animals. Tissue used from the microarray studies analysed in this letter came from a variety of joints from immature mice and are likely to include mixed chondrocyte phenotypes. Where stated explicitly, chondrocytes were expanded in monolayer cultures following collagenase based isolation (E-GEOD-8052 and E-GEOD-7683). All 3 studies (E-GEOD-10556, E-GEOD-18052 and E-GEOD-7683) used the same Affymetrix Mouse430_2 chips. (D) Commonality between rat derived datasets. The rat femoral head (E-GEOD-6119, E-GEOD-14402) or knee (E-GEOD-8077) tissue was harvested from a range of ages from one day old neonates (from which “only the outer two-thirds of cartilage” was used to select for articular chondrocytes, E-GEOD-14402) to several month old rats (300-320g, E-GEOD-8077). Strain was either Wistar (E-GEOD-6119), Sprague-Dawley (E-GEOD-8077) or not stated. E-GEOD-6119 and E-GEOD-14402 both used monolayer expanded chondrocytes following collagenase II based isolation. E-GEOD-6119 also included pronase, but E-GEOD-8077 used direct RNA extraction from macerated tissue. All the included rat studies used the Affymetrix Rat230_2 chips. One bovine dataset derived from chondrocytes 3D cultured from carpal bones of 3 to 6 mo old calves was also analyzed (E-GEOD-18394, Affymetrix Bovine chip, annotated with version “na29”). Since there was only 1 bovine chondrocyte dataset on EBI (albeit including a number of replicates) this is not included in the Venn diagrams. All other datasets were annotated with revision Affymetrix annotation version “na31”. Note that each of the 3 species sets in (A) is equivalent to the commonly expressed regions of the Venn diagrams in (B, C, and D).
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Figure 1: Figure 1. Commonality of gene transcript expression between datasets. (A) Commonality between species. These represent transcripts present in each of the 3 datasets for each of the human, mouse, and rat datasets (i.e., includes 9 datasets total). Far more are observed in rodent datasets (mouse especially) than human. (B) Commonality between human derived datasets. The human studies used tissue harvested from either adolescents receiving limb length correction surgery (E-GEOD-1277), adults receiving ACL surgery (E-GEOD-16464), or post mortem (E-GEOD-10024). Samples were taken from knee (E-GEOD-16464), distal femur (E-GEOD-1277), or shoulder (E-GEOD-10024) and focussed on articular (E-GEOD-10024), mixed (E-GEOD-16464), or growth plate (E-GEOD-1277) chondrocytes. Chip Ids; E-GEOD-10024 used HG-U133A and E-GEOD-16464 used the slightly newer HG-U133A_Plus_2, but E-GEOD-1277 used the U95AV2 GeneChip. All 3 human studies used expanded chondrocytes, but E-GEOD-10024 and E-GEOD-16464 re-constituted those into 3D cultures. Extraction enzymes were collagenase P (E-GEOD-10024), clostridial collagenase and deoxyribonuclease I (E-GEOD-16464) and trypsin (E-GEOD-1277). (C) Commonality between mouse derived datasets. Rodent studies suffer from inherent difficulties in extraction of tissue since cartilage is thinner than larger animals. Tissue used from the microarray studies analysed in this letter came from a variety of joints from immature mice and are likely to include mixed chondrocyte phenotypes. Where stated explicitly, chondrocytes were expanded in monolayer cultures following collagenase based isolation (E-GEOD-8052 and E-GEOD-7683). All 3 studies (E-GEOD-10556, E-GEOD-18052 and E-GEOD-7683) used the same Affymetrix Mouse430_2 chips. (D) Commonality between rat derived datasets. The rat femoral head (E-GEOD-6119, E-GEOD-14402) or knee (E-GEOD-8077) tissue was harvested from a range of ages from one day old neonates (from which “only the outer two-thirds of cartilage” was used to select for articular chondrocytes, E-GEOD-14402) to several month old rats (300-320g, E-GEOD-8077). Strain was either Wistar (E-GEOD-6119), Sprague-Dawley (E-GEOD-8077) or not stated. E-GEOD-6119 and E-GEOD-14402 both used monolayer expanded chondrocytes following collagenase II based isolation. E-GEOD-6119 also included pronase, but E-GEOD-8077 used direct RNA extraction from macerated tissue. All the included rat studies used the Affymetrix Rat230_2 chips. One bovine dataset derived from chondrocytes 3D cultured from carpal bones of 3 to 6 mo old calves was also analyzed (E-GEOD-18394, Affymetrix Bovine chip, annotated with version “na29”). Since there was only 1 bovine chondrocyte dataset on EBI (albeit including a number of replicates) this is not included in the Venn diagrams. All other datasets were annotated with revision Affymetrix annotation version “na31”. Note that each of the 3 species sets in (A) is equivalent to the commonly expressed regions of the Venn diagrams in (B, C, and D).

Mentions: It should be noted that these microarray datasets were derived from different species (3 rat, 3 mouse, 3 human and 1 bovine) and there are potential differences in chondrocyte isolation protocols. Constraining analysis to just rodent (6 datasets) returns a set of 23 commonly expressed ion channel genes (Table 2). Figure 1 quantitatively illustrates both the overlap of genes commonly expressed between species (Fig. 1A) and the overlap between each of the transcripts from human, mouse and rat (Fig. 1B, C, and D, respectively). It is evident that far more transcripts were detected in all 3 of the mouse datasets than in all 3 of the human datasets. This could be for three reasons; firstly, it is possible that the sensitivity of the mouse chips is greater, but we have seen no specific evidence for this. Secondly, each of the protocols requires manual dissection and separation of chondrocytes from the subchondral bone and adnexa. It is possible that mouse “chondrocyte” samples are inherently contaminated with non-chondrocyte tissue. In an electrophysiological or immunohistochemical study such contamination would be relatively easy to detect, but in a biochemical protocol, where harvested tissue is macerated and then processed, it could be missed. Thirdly, it is conceivable that there are genuine phenotypic differences between chondrocytes in mice and other animals. Such differences have been discussed elsewhere.22,23


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 1. Commonality of gene transcript expression between datasets. (A) Commonality between species. These represent transcripts present in each of the 3 datasets for each of the human, mouse, and rat datasets (i.e., includes 9 datasets total). Far more are observed in rodent datasets (mouse especially) than human. (B) Commonality between human derived datasets. The human studies used tissue harvested from either adolescents receiving limb length correction surgery (E-GEOD-1277), adults receiving ACL surgery (E-GEOD-16464), or post mortem (E-GEOD-10024). Samples were taken from knee (E-GEOD-16464), distal femur (E-GEOD-1277), or shoulder (E-GEOD-10024) and focussed on articular (E-GEOD-10024), mixed (E-GEOD-16464), or growth plate (E-GEOD-1277) chondrocytes. Chip Ids; E-GEOD-10024 used HG-U133A and E-GEOD-16464 used the slightly newer HG-U133A_Plus_2, but E-GEOD-1277 used the U95AV2 GeneChip. All 3 human studies used expanded chondrocytes, but E-GEOD-10024 and E-GEOD-16464 re-constituted those into 3D cultures. Extraction enzymes were collagenase P (E-GEOD-10024), clostridial collagenase and deoxyribonuclease I (E-GEOD-16464) and trypsin (E-GEOD-1277). (C) Commonality between mouse derived datasets. Rodent studies suffer from inherent difficulties in extraction of tissue since cartilage is thinner than larger animals. Tissue used from the microarray studies analysed in this letter came from a variety of joints from immature mice and are likely to include mixed chondrocyte phenotypes. Where stated explicitly, chondrocytes were expanded in monolayer cultures following collagenase based isolation (E-GEOD-8052 and E-GEOD-7683). All 3 studies (E-GEOD-10556, E-GEOD-18052 and E-GEOD-7683) used the same Affymetrix Mouse430_2 chips. (D) Commonality between rat derived datasets. The rat femoral head (E-GEOD-6119, E-GEOD-14402) or knee (E-GEOD-8077) tissue was harvested from a range of ages from one day old neonates (from which “only the outer two-thirds of cartilage” was used to select for articular chondrocytes, E-GEOD-14402) to several month old rats (300-320g, E-GEOD-8077). Strain was either Wistar (E-GEOD-6119), Sprague-Dawley (E-GEOD-8077) or not stated. E-GEOD-6119 and E-GEOD-14402 both used monolayer expanded chondrocytes following collagenase II based isolation. E-GEOD-6119 also included pronase, but E-GEOD-8077 used direct RNA extraction from macerated tissue. All the included rat studies used the Affymetrix Rat230_2 chips. One bovine dataset derived from chondrocytes 3D cultured from carpal bones of 3 to 6 mo old calves was also analyzed (E-GEOD-18394, Affymetrix Bovine chip, annotated with version “na29”). Since there was only 1 bovine chondrocyte dataset on EBI (albeit including a number of replicates) this is not included in the Venn diagrams. All other datasets were annotated with revision Affymetrix annotation version “na31”. Note that each of the 3 species sets in (A) is equivalent to the commonly expressed regions of the Venn diagrams in (B, C, and D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Figure 1. Commonality of gene transcript expression between datasets. (A) Commonality between species. These represent transcripts present in each of the 3 datasets for each of the human, mouse, and rat datasets (i.e., includes 9 datasets total). Far more are observed in rodent datasets (mouse especially) than human. (B) Commonality between human derived datasets. The human studies used tissue harvested from either adolescents receiving limb length correction surgery (E-GEOD-1277), adults receiving ACL surgery (E-GEOD-16464), or post mortem (E-GEOD-10024). Samples were taken from knee (E-GEOD-16464), distal femur (E-GEOD-1277), or shoulder (E-GEOD-10024) and focussed on articular (E-GEOD-10024), mixed (E-GEOD-16464), or growth plate (E-GEOD-1277) chondrocytes. Chip Ids; E-GEOD-10024 used HG-U133A and E-GEOD-16464 used the slightly newer HG-U133A_Plus_2, but E-GEOD-1277 used the U95AV2 GeneChip. All 3 human studies used expanded chondrocytes, but E-GEOD-10024 and E-GEOD-16464 re-constituted those into 3D cultures. Extraction enzymes were collagenase P (E-GEOD-10024), clostridial collagenase and deoxyribonuclease I (E-GEOD-16464) and trypsin (E-GEOD-1277). (C) Commonality between mouse derived datasets. Rodent studies suffer from inherent difficulties in extraction of tissue since cartilage is thinner than larger animals. Tissue used from the microarray studies analysed in this letter came from a variety of joints from immature mice and are likely to include mixed chondrocyte phenotypes. Where stated explicitly, chondrocytes were expanded in monolayer cultures following collagenase based isolation (E-GEOD-8052 and E-GEOD-7683). All 3 studies (E-GEOD-10556, E-GEOD-18052 and E-GEOD-7683) used the same Affymetrix Mouse430_2 chips. (D) Commonality between rat derived datasets. The rat femoral head (E-GEOD-6119, E-GEOD-14402) or knee (E-GEOD-8077) tissue was harvested from a range of ages from one day old neonates (from which “only the outer two-thirds of cartilage” was used to select for articular chondrocytes, E-GEOD-14402) to several month old rats (300-320g, E-GEOD-8077). Strain was either Wistar (E-GEOD-6119), Sprague-Dawley (E-GEOD-8077) or not stated. E-GEOD-6119 and E-GEOD-14402 both used monolayer expanded chondrocytes following collagenase II based isolation. E-GEOD-6119 also included pronase, but E-GEOD-8077 used direct RNA extraction from macerated tissue. All the included rat studies used the Affymetrix Rat230_2 chips. One bovine dataset derived from chondrocytes 3D cultured from carpal bones of 3 to 6 mo old calves was also analyzed (E-GEOD-18394, Affymetrix Bovine chip, annotated with version “na29”). Since there was only 1 bovine chondrocyte dataset on EBI (albeit including a number of replicates) this is not included in the Venn diagrams. All other datasets were annotated with revision Affymetrix annotation version “na31”. Note that each of the 3 species sets in (A) is equivalent to the commonly expressed regions of the Venn diagrams in (B, C, and D).
Mentions: It should be noted that these microarray datasets were derived from different species (3 rat, 3 mouse, 3 human and 1 bovine) and there are potential differences in chondrocyte isolation protocols. Constraining analysis to just rodent (6 datasets) returns a set of 23 commonly expressed ion channel genes (Table 2). Figure 1 quantitatively illustrates both the overlap of genes commonly expressed between species (Fig. 1A) and the overlap between each of the transcripts from human, mouse and rat (Fig. 1B, C, and D, respectively). It is evident that far more transcripts were detected in all 3 of the mouse datasets than in all 3 of the human datasets. This could be for three reasons; firstly, it is possible that the sensitivity of the mouse chips is greater, but we have seen no specific evidence for this. Secondly, each of the protocols requires manual dissection and separation of chondrocytes from the subchondral bone and adnexa. It is possible that mouse “chondrocyte” samples are inherently contaminated with non-chondrocyte tissue. In an electrophysiological or immunohistochemical study such contamination would be relatively easy to detect, but in a biochemical protocol, where harvested tissue is macerated and then processed, it could be missed. Thirdly, it is conceivable that there are genuine phenotypic differences between chondrocytes in mice and other animals. Such differences have been discussed elsewhere.22,23

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