Osteocyte_Osteoblast_Mechanical

MIAME is the abbreviation of "Minimum Information About a Microarray Experiment", (www.mged.org/miame) which is established by Microarray Gene Expression Data (MGED) Society. MGED is an international organization of microarray reserchers (biologists, computer scienticsts, stasticians) that are developing microarray data quality standards to promote the sharing of microarray resources generated by functional genomics and proteomics high throughput study.

Nature and the Lancet regard MIAME checklist as a requirement for publishing microarray papers.

MIAME CHECKLIST

MGED Guide		Notes	Manuscript	Supplementary website
Experiment Design	Type of Experiment	1) osteoblasts vs. osteocytes; 2) osteoblasts or osteocytes at different cell-communication status; 3) osteoblasts or osteocytes subjected to fluid flow; 4) osteoblasts vs. osteocytes when subjected to fluid flow. (More details ...)
	Experimental factors	1) cell-communication status; 2) fluid flow; 3) short or long term after fluid flow. (More details ...)
	The number of hybridizations performed	Triplicate hybridization for each experimental condition. (More details ...)
	The type of reference used for the hybridizations	(If any)
	Hybridization design	(If applicable) a description of the comparisons in each hybridization -- to a standard reference sample? Between experimental sammples? (diagram or table may be useful)
	Quality control steps taken	1) For each experimental factors: two or three replication of hybridization performed; 2) For each chip: housekeeping genes, cDNA synthesis control spots, negative controls (phage λ DNA).
	URL of supplemental websites / database
Samples	The origin / characteristics of the biological samples	1) 2T3 osteoblast-osteocyte differentiation cell model (from transgenic mouse containing BMP-2 promoter driving the SV-40 T antigen transgene; 2) MLO-Y4 osteocyte-like cell model (from mouse containing osteocalcin promoter driving T-antigen). (More details ...)
	Manipulation of biological samples & protocols	1) 2T3 was cultured in α-MEM with 10% FBS; 2) MLO-Y4 was cultured in α-MEM with 5% FBS and 5% CS on collagen -coated surfaces; 3) fluid flow. (More details ...)
	Protocols for preparing the hybridization extracts	1) Cells were lysed in RNA-Bee(Tel-test), then the RNA was obtained from chloroform separation, precipitated by isopropanol and washed with ethanol; 2) Poly-A+ RNA; 3) Purification protocol
	Labeling protocols
	External controls
Hybridization procedures & parameters	Hybridization
	Blocking
	Washing
Measurement data & specifications	Type of scanning hardware	Clontech (BD Biosciences, Palo Alto, CA.). (More details ...)
	Type of scanning software	Packard Cyclone Phosphoimager using Super Resolution Type SR screen Optiquant. (More details ...)
	Type of image analysis software	AtlasImage 2.7beta (BD Biosciences, Palo Alto, CA.). (More details ...)
	A description of the measurements produced by the image-analysis software	16-bit super resolution phosphorimaging tif files. (More details ...)
	A description of the measurements used in the analysis
	The complete output of the image analysis (spot quantitation matrices)	(link)
	Data selection / transformation procedures	(link)
	Gene expression data tables after normalization	(link)
	Final gene expression data tables based on that the authors make the conclusions	(link)
Array Design	General	Platform type? Surface/Coating specifications? The availability of the array?
	Each feature (spot) on the array	Its location on the array? ID of its respective reporter?
	Each reporter	Its type? Its information / characteristics -- database references, sequence (if available)?
	Commercial arrays	Manufacturer information -- catalogue#; website?

Experiment Design

Type of experiment:

For our microarray experiments, we investigate the answers of the questions:

1. How gene expression differs between osteoblasts and osteocytes?
2. How gene expression changes when osteoblasts or osteocytes are at different cell-communication status?
3. How gene expression changes when osteoblasts or osteocytes are subjected to fluid flow?
3. How gene expression changes differ between osteoblasts and osteocytes when they are both subjected to fluid flow?

(Details as intepreted in the paper)

Experimental factors:

(Details as intepreted in the method part of the paper)

The number of hybridizations performed:

Triplicate hybridization for each experimental condition.

(Details as intepreted in the method part of the paper)

The type of reference used for the hybridizations:

Hybridization design:

Quality control steps taken:

URL of supplemental website:

Samples

The origin / characteristics of the biological sample:

2T3 osteoblast-osteocyte differentiation cell model

• Isolated and cloned from a transgenic mouse containing BMP-2 promoter driving the SV-40 T antigen transgene and well-characterized previously.

• Represents pre-osteoblast that can differentiate into osteocyte like cells.

• Produce a mineralized bone-like matrix.

• Respond to bone forming agents, such as Bone Morphogenetic Proteins (BMP).

• The structure and mineral composition of the mineralized bone matrix produced by 2T3 cells is similar to the normal woven bone and the mineralized bone matrix produced by primary cultures of FRC osteoblasts

MLO-Y4 osteocyte-like cell model

• Derived from a mouse containing the osteocalcin promoter driving T-antigen.

• Represents an early pre-osteocyte stage that is not in a mineralized matrix and still has the capacity to grow.

• Produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural/marrow/osteocyte antigen CD44, and for connexin 43, a protein found in gap junctions.

• Produces very small amounts of type I collagen mRNA compared with primary osteoblasts

Manipulation of biological samples & protocols:

2T3 cells were cultured in α-MEM supplemented with 10% FBS. The cells were incubated in a 5% CO2 incubator at 37˚C

MLO-Y4 cells were grown in α -MEM supplemented with 5% FBS and 5% CS. The cells were incubated in a 5% CO2 incubator at 37˚C and cultured on

collagen -coated (rat tail collagen type I, 0.15 mg/ml) surfaces.

Mechanical loading protocols as intepreted in the method part of the paper.

Protocols for preparing the hybridization extracts:

Labeling protocols:

External controls :

Hybridization procedures & parameters

Hybridization

Blocking

Washing

Measurement data and specifications

Type of scanning hardware

Type of scanning software

Type of image analysis software

A description of the measurements produced by the image-analysis software

A description of the measurements used in the analysis

The complete output of the image analysis (spot quantitation matrices)

Data selection / transformation procedures

Gene expression data tables after normalization

Final gene expression data tables based on that the authors make the conclusions

Array design

General

Each feature (spot) on the array

Each reporter

Commercial arrays