Minimum information about a microarray experiment

Minimum information about a microarray experiment - MIAME

Draft March 21, 2001 (based on November 17, 2000) updated 30 July 2001.

MIAME compliant example experiment

1. Experimental design: the set of the hybridisation experiments as a whole

This section gives information describing the experiment, which may consist of one or more hybridisations, as a whole. Normally 'experiment' should include a set of hybridisations which are inter-related and performed in a limited period of time. For instance, it may be all the hybridisations related to research published in a single paper.

author (submitter), laboratory, contact information, links (URL) -

a) author (submitter), laboratory, contact information, links (URL), citation

T. Preiss

E. Wabek

A. Richter

J. Zimmermann

M. Wagner

C. Schwager

M. W. Hentze

W. Ansorge

EMBL, Meyerhofstr.1, D-69117 Heidelberg, Germany

b) type of the experiment - maximum one line

treated vs. untreated comparison

c) experimental variables, i.e. parameters or conditions tested (e.g., time, dose, genetic variation, response to a treatment or compound)

response to a compound - rapamycin

d) single or multiple hybridisations

For multiple hybridisations:

* serial (yes/no)

* grouping(yes/no)

for multiple hybridisations:

* serial (yes/no)

* type (e.g., time course, dose response)

dose response

grouping (yes/no)

* type (e.g., normal vs. diseased, multiple tissue comparison)

pre and post compound addition

Relationships between all the samples, arrays and hybridisations in the experiment: each sample, each array, and each hybridisation should be given a unique ID or number, and all the relationships should be listed, possibly with appropriate comments.

Samples: treated, untreated

Arrays: 20, 27, 28, 29, nn

Hybridisations: H1: Array 20, treated Cy5 vers. untreated Cy3

H2: Array 27, treated Cy5 vers. untreated Cy3

H3: Array 28, treated Cy5 vers. untreated Cy3

H4: Array 29, treated Cy5 vers. untreated Cy3

H5: Array nn, treated Cy5 vers. untreated Cy3

H1 and H3 are replicates

All are replicates to each other,

same RNA prep was used for all hybridisations

cDNA labelling was done individually for each experiment

e) quality related indicators quality control steps taken:

* biological replicates

* technical replicates (replicate spots or hybs)

* polyA tails

* low complexity regions

* unspecific binding

* other

Empty wells, labeled primers, spiked in controls from Arabidopsis and bacterial genes, postive as well as negative control sequences

f) optional user defined "qualifier, value, source" list

g) a free text description of the experiment set or a link to a publication

The diploid yeast (Saccharomyces cerevisiae) strain FY1679 (Mata/? ura3-52/ura3-52 trp1?63/+leu2?/+his3?200/+) was grown at 30 °C in YPD medium.

At an OD(600 nm) of 0.6 rapamycin was added to a final concentration of 0.2 µg/ml and incubation was continued for 90 min.

A control culture was treated in parallel with an equal amount of DMSO vehicle.

Cells were harvested at an OD(600nm) of 0.9 (+rapamycin) and 1.1 (control).

Unpublished data.

There are three published papers describing the transcriptional programme of yeast in response to rapamycin.

It should be possible to compare our datasets with those described in these papers:

Curr. Biol. 2000 10:1574-1581;

PNAS USA 1999 96:14866-14870;

Genes Dev. 1999 13:3271-3279

2. Array design: each array used and each element (spot) on the array

There are two parts of this section:

2.1 describes the list of physical arrays themselves, each

of these referring to specific array design types described in 2.2. We expect that the array design type

descriptions will be given by the array providers and manufactures, in which case the users will simply

need to reference them.

2.2 Array design This section consists of three parts

a) description of the array as the whole,

b) description of each type of elements (spot) used (properties that are typically common to many elements

(e.g., 'synthesized oligo-nucleotides' or 'PCR products from cDNA clones'),

c) description of the specific properties of each element, such as the DNA sequence.

(In practice, the last part will be provided as a spread-sheet or tab-delimited file)

2.1 Array copy: each array used and each element (spot) on the array.

* unique id as used in part 1

20,27,28,29

* array design name (e.g Stanford human 10K set)

(for commercial or standard arrays a unique ID given by the provider may be used)

EMBL Yeast 12K ver.1

2.2

a) array related information

* array design name (e.g., "Stanford Human 10K set")

EMBL Yeast 12KVer. 1

* platform type: in situ synthesized or spotted

spotted

* array provider (source)

in-house (EMBL)

* surface type: glass, membrane, other

glass

* surface type name

in-house coated EMBL slides

* physical dimensions of array support (e.g. slide)

75 x 25 mm

* number of elements on the array

14 000 (approximately)

* a reference system allowing to locate each element (spot) on the array

(in the simplest case the number of columns and rows is sufficient)

by coordinate, referencing to an external data table,

32 subgrids

ordered 4 x8

counted left right, top down, 21 rows, 20 columns per subgrid,

counted left right, top down for array EMBL Yeast 12KVer. 1

* production date

* production protocol (obligatory if applicable)

Yeast cDNA microarrays were spotted using pcr products obtained with

the Research Genetics GenePair primer set.

Spotters: GeneMachine Omnigrid, Telechem split pins SMP3

Double stranded, preferentially only the forward strand is attached

via a 5' aminolink moiety.

(The slide chemistry will preferentially bind this strand but

it will also retain the other).

* optional "qualifier, value, source" list (see Introduction)

b)properties of each group of elements (spots) on the array;

elements may be simple, i.e., containing only identical molecules, or composite, i.e., containing different oligonucleotides obtained from the same reference molecule;

* element type id

* simple or composite

simple

* element type: synthesized oligo-nucleotides, PCR products, plasmids, colonies, other

PCR products

* single or double stranded

double

* element (spot) dimensions (approximate diameter)

90-120µm

* element generation protocol that includes sufficient information to reproduce the element

using the ResGen primer set and genomic DNA

(some PCR products encompass intron sequences)

* attachment (covalent/ionic/other)

covalent

* optional "qualifier, value, source" list (see Introduction)

c) specific properties of each spot on the array:

* element type ID from 2.2b

PCR products

* position on the array allowing spot identification in the image (see 5a below)

spreadsheet attached

* clone information, obligatory for elements obtained from clones:

(clone ID,clone provider,date,availability )

* sequence or PCR primer information:

(sequence accession number in DDBJ/EMBL/GenBank if known, sequence itself (if databases do not contain it, primer pair information, if relevant)

The PCR primers that are used for the generating the elements are

identified by MIPS ORF names given in attached spreadsheet

* for composite oligonucleotide elements:

(oligonucleotide sequences if given,given number of oligonucleotides and the reference sequence (or accession number), otherwise, one of the above should unambiguously identify the element.

* approximate lengths if exact sequence not known

* gene name and links to appropriate databases

e.g., SWISS-PROT, or organism specific databases), if known and relevant

(Normally this information will be provided in one or more spread-sheets or tab-delimited files. )

3. Samples: samples used, extract preparation and labeling

By a 'sample' we understand the biological material, from which the RNA gene products (or DNA) have been extracted for subsequent labeling, hybridisation and measuring. This section describes the source of the sample (e.g., organism, cell type or line), its treatment, as well as preparing the extract and its labeling, i.e., all steps that precedes the contact with an array (i.e., hybridisation). Each sample used in the experiment has a separate section 3. In practice, if the treatments are similar, differing only slightly, the descriptions can be given together, clearly pointing out the differences.

sample source and treatment (this section describes the biological treatment which happens before the extract preparation and labelling, i.e., biological sample in which we intend to measure the gene expression; for each sample only some of the qualifiers given below may be relevant):

* ID as used in section 1

treated, untreated

* organism (NCBI taxonomy)

Saccharomyces cerevisiae

additional "qualifier, value, source" list; each qualifier in the list is obligatory if applicable; the list includes:

* cell source and type (if derived from primary sources (s))

strain FY1679

* development stage

diploid

* genetic variation (e.g., gene knockout, transgenic variation)

Mata/? ura3-52/ura3-52 trp1?63/+leu2?/+his3?200/+

* in vivo treatments (organism or individual treatments)

* in vitro treatments (cell culture conditions)

cells were grown at 30^oC in YPD medium

* treatment type (e.g., small molecule, heat shock, cold shock, food deprivation)

small molecule

* compound

rapamycin, drug vehicle

* separation technique (e.g., none, trimming, microdissection, FACS)

none

* laboratory protocol for sample treatment

At an OD(600 nm) of 0.6 rapamycin (20µl of a 1 µg/µl solution in DMSO)

was added to a final concentration of 0.2 µg/ml and incubation was continued

for 90 min. A control culture was treated in parallel with 20µl of DMSO

vehicle. Cells were harvested at an OD(600nm) of 0.9 (+rapamycin) and 1.1 (control).

b) hybridisation extract preparation laboratory protocol for extract preparation, including:

protocol description

* Description:

Total RNA preparation from yeast cultures

Grow 100ml suspension culture in YPD to an OD @600nm of approx 1

Harvest cells by centrifugation for 10' @5000rpm/4°C in the SS34 rotor

Resuspend cells in breaking buffer and transfer to 2 x 2ml eppies

per culture

Pellet cells 2' @ top speed in a cooled microfuge

Freeze pellet in liquid nitrogen or proceed directly to cell lysis

To cell pellet add 400µl phenol/chloroform,

400µl breaking buffer, 5µl 20% SDS, and approx 400µl glass beads

Agitate 2x for 45'' in a carbon dioxide-cooled bead mill

(GeneExProgramme) taking care not to freeze the mixture

Spin 10' @ top speed in a cooled microfuge

Remove SN to a new eppie

Perform 2x regular phenol/chloroform extractions

Precipitate with ethanol/ sodium acetate as usual

Materials:

Breaking buffer:

20mM Tris/Cl pH7.4, 100mM KCl, 2mM MgCl2, 2mM DTT

glass beads Sigma # G-8772

* extraction method

bead beating lysis

* whether total RNA, mRNA, or genomic DNA is extracted

total RNA

* amplification (RNA polymerases, PCR)

none

* optional "qualifier, value, source" list (see Introduction)

c)labelling: laboratory protocol for labelling, including:

* protocol

Clontech Atlas Glass Fluorescent labeling kit was used

(with some modifications).

cDNA synthesis was primed with oligo(dT)20 primer.

* amount of nucleic acids labeled

20 (g total RNA) ????

* label used (e.g., Cy3, Cy5, 33P)

Cy3 (untreated), Cy5 (treated)

* optional "qualifier, value, source" list (see Introduction)

4. Hybridisations: procedures and parameters

This section describes details of each hybridisation in the experiment.

Each hybridisation has a separate section 4, though if they are similar they may be described together.

* ID as given in section 1

H1, H2, H3, H4, H5

* laboratory protocol for hybridisation, including:

* the solution (e.g., concentration of solutes)

Hybridisation buffer:

50% formamide, 6x SSC, 0.5% SDS, 5x Denhardt's solution.

* blocking agent

6x SSC, 0.5% SDS, 1% BSA. Prehybridisation for 45 min at 42°C.

* Slide blocking:

6x SSC, 0.5% SDS, 1% BSA. Prehybridisation for 45 min at 42°C.

* Probe blocking:

0.1 ug/ul salmon sperm DNA, 0.5 ug/ul polydA during hybridization.

* wash procedure

1x 10 min wash with 0.1x SSC, o.1% SDS, 2x 5 min in 0.1x SSC

* quantity of labelled target used

all material generated from 20 µg total RNA

* time, concentration, volume, temperature

overnight (16h), 40 µl, at 42°C

* description of the hybridisation instruments

Gene Machine Hybridisation chamber

* optional "qualifier, value, source" list (see Introduction)

5. Measurements: images, quantitation, specifications:

This section describes the data obtained from each scan and their combinations

hybridisation scan raw data:

a1) the scanner image file (e.g., TIFF) from the hybridised microarray scanning

attached files

AXON_27_Treated_Cy5.tif

AXON_27_Untreated_Cy3.tif

AXON_28_Treated_Cy5.tif

AXON_28_Untreated_Cy3.tif

AXON_29_Treated_Cy5.tif

AXON_29_Untreated_Cy3.tif

GMS_27_Treated_Cy5.tif

GMS_27_Untreated_Cy3.tif

GMS_20_Treated_Cy5.tif

GMS_20_Untreated_Cy3.tif

GMS_29_Treated_Cy5.tif

GMS_29_Untreated_Cy3.tif

GSI_27_Treated_Cy5.tif

GSI_27_Untreated_Cy3.tif

GSI_20_Treated_Cy5.tif

GSI_20_Untreated_Cy3.tif

GSI_nn_Treated_Cy5.tif

GSI_nn_Untreated_Cy3.tif

Grid Files:

==========

AXON_27_***.grid

AXON_28_***.grid

AXON_29_***.grid

GMS_20_***.grid

GMS_27_***.grid

GMS_28_***.grid

GSI_20_***.grid

GSI_27_***.grid

GSI_nn_***.grid

ii.a2) scanning information:

* parsed header of the TIFF file, including laser power, spatial resolution, pixel space, PMT voltage;

see attached files

* laboratory protocol for scanning, including:

* hybridisation ID as in Section 1

H1, H2, H3, H4, H5

* image unique id

* scanning parameters (including laser power,spatial resolution,pixel space PMT voltage)

* lab protocol for scanning (including scanning hardware and software)

* scanning hardware

Genetic Microsystems GMS 418 (H1, H2, H4)

Axon GenePix4000A (H2, H3, H4)

GSI Lumonics ScanArray 5000 (H1, H2,H5)

* scanning software

b) image analysis and quantitation

bi) the complete image analysis output (of the particular image analysis software) for each element (or composite element - see 2.b)),

for each channel -

see attached files:

Table Data Files:

=============

AXON_27.txt

AXON_28.txt

AXON_29.txt

GMS_20.txt

GMS_27.txt

GMS_28.txt

GSI_20.txt

GSI_27.txt

GSI_nn.txt

bii) image analysis information:

* input image id

* quantitation unique id

* image analysis software specification and version, availability, and the description of the algorithm

ChipSkipper EMBL Christian Schwager (schwager@embl-heidelberg.de)

* all parameters

Expected spot diameter: 100 Micrometer (11pixel)

Area reject = 10

C) summarized information from possible replicates

ci) derived measurement value summarizing related elements as used by the author (this may constitute replicates of the element on the same or different arrays or hybridisations, as well as different elements related to the same entity e.g. gene)

attached files

Average Files (Scanner specific averages)

===============================

AXON_Average.txt

AXON_Average.xls

GMS_Average.txt

GMS_Average.xls

GSI_Average.txt

GSI_Average.xls

cii) reliability indicator for the value of c1) as used by the author (e.g. standard deviation); may be "unknown"

see files above

ciii) specification how c1 and c2 are calculated; the specification should be bases on b1

see files above

6. Normalisation controls, values, specifications for hybridisations

a) Normalization strategy (spiking, housekeeping genes, total array)

spiking

b) Normalisation algorithm (linear regression, log-linear regression, ratio statistics, log(ratio) mean median centering)

linear regression

c) Control array elements

* position (the abstract coordinate on the array)

attached spreadsheet

* control type (spiking, normalization, negative, positive)

spiking

* control qualifier (endogenous, exogenous)

exogenous

d) Hybridisation extract preparation

* spike type

polyadenylated spiking mRNA added prior to reverse transcription

* target element

* optional user defined quality value

(to be added as section 7 in the next MIAME version)

List of the control elements used on the EMBL yeast chips:

All fragments have been amplified by PCR using Research genetics

style primers, i.e. they all have the same short invariant regions

added by the primer as the yeast orfs.

The sequences shown below show only the "gene"-specific part.

The specific part of the primers that were used to amplify these

fragments are the 20 or so nucleotides at the extremes of the listed sequences.

The following sequences were employed as spiking controls:

P450, FAD6, 6i18, 15b8, 8h10, CAT, LUC

All other control spots were assumed to be negative controls.

Linear regression to compensate between the two channels was done

using all control spots (negative and positive).

The chips also contained repeated spots of fluorescently labelled oligonucleotides named "Cy3" or "Cy5".

These spots were not included in any data evaluation but are also listed in the data tables.