Investigation Title Transcription profiling of left and right lateral plates of mouse 8.5dpc embryos to identify assymetrically expressed genes Comment[Submitted Name] ID of left-right asymmetrically expressed genes in mouse 8.5dpc embryos Experimental Design is_expressed_design co-expression_design dye_swap_design transcription profiling by array transcription profiling by array Experimental Design Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology EFO EFO Comment[SecondaryAccession] Comment[ArrayExpressReleaseDate] 2010-06-29 Comment[AEMIAMESCORE] 2 Comment[ArrayExpressAccession] E-MEXP-2277 Comment[MAGETAB TimeStamp_Version] 2010-08-10 11:48:34 Last Changed Rev: 13058 Experimental Factor Name ORGANISM_PART Experimental Factor Type organism_part Experimental Factor Term Source REF Person Last Name microarray Person First Name MGU Person Mid Initials Person Email d.williams@har.mrc.ac.uk Person Phone 01235 841135 Person Fax Person Address Harwell, didcot, OXON, ox11 0rd, United Kingdom Person Affiliation MRC Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-06-29 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description C3H/HeH females were mated with 101/H males

Mating was assessed by the presence of vaginal plugs the next morning. Identification of plug was designated day 0.5.



At 8.5 days of development the dam was culled by cervical dislocation, the uterine horns disscted and placed into PBS. The uterus was dissected to release decuduae and these opened according to standard protocols. Morphologically normal looking embryos of between 4 and 6 somites were collected. The left and right lateral plates were dissected away with watchmakers forceps and pools of 4 left and 4 right lateral plates were snap frozen in LN2. Protocol Name P-MTAB-4894 P-MTAB-4899 P-MTAB-4898 P-MTAB-4896 P-MTAB-4893 P-MTAB-4900 P-MTAB-4897 Protocol Type nucleic_acid_extraction specified_biomaterial_action grow pool labeling labeling hybridization Protocol Description For 100 mg, 500 mg or 1.0 g tissue;



1. Grind tissue to powder under dry ice (or liquid N2)

Add to GITC solution D (i.e. with B-mercaptoethanol)



100 mg 500 mg 1 g

Solution D 1 mL 5 mL 10 mL



Mix well & homogenise tissue solution using syringe + 18G needle.



2. Add the following to each sample;

100 mg 500 mg 1 g

2M NaOAc (pH 4.0) 50 uL 250 uL 500 uL

Aqueous phenol 500 uL 2.5 mL 5.0 mL

Chloroform-IAA 100 uL 500 uL 1 mL



Mix well & leave on wet ice x 15 mins.

Spin in centrifuge x full speed x 15 mins @ 4 OC



3. Remove top layer* to a clean tube (RNase-free) & add equal volume of cold isopropanol.

* If this homogenate is very viscous, repeat the aq.phenol-chloroform step (2).



Mix well & leave @ –20 OC x 15 - 30* mins (45 mins max. or will bring out impurities).



Spin x full speed x 15 mins @ 4 OC



4. Remove supernatant carefully & should see a white RNA pellet.

Add the following;

100 mg 500 mg 1 g

Solution D 100 uL 500 uL 1 mL

Isopropanol (cold) 100 uL 500 uL 1 mL



Mix well & leave at –20 OC x 15 mins.

Spin x full speed x 10 mins @ 4 OC.





5. Remove supernatant carefully & briefly air-dry RNA pellet.

Add the following;

100 mg 500 mg 1 g

75% ethanol (in RNase-free H20) 800 uL 2 mL 4 mL



6. Mix well & leave at –20 OC for at least 15 mins (i.e. as long as is convenient!)

Spin x full speed x 5 mins @ 4 oC.



7. Remove ethanol & air-dry RNA pellet x ~ 5 mins at room temperature.

Do not let pellet dry out completely or it will be difficult to resuspend!



8. Resuspend pellet in as small a volume of H2O (RNase-free) as possible – it may need heating to 65 oC x 5 mins to resuspend completely.

100 mg 500 mg 1 g

H2O (RNase-free) 50 uL 200 uL 400 uL



9. Add 1 uL/ 2 uL / 5 uL RNase inhibitor to each sample.



10. Remove 2-5 uL for OD measurements.



11. Run ~ 5 ug RNA out on 2% agarose gel to check quality (18/28s bands).



1) Place the extracted RNA in a suitably sized vessel for the Rotor–stator homogenizer (e.g., in a 10–15 ml tube). Add the appropriate volume of Buffer RLT (see Table 1 below). Homogenize immediately using a conventional rotor–stator homogenizer for 45 s to 60 s until the sample is uniformly homogeneous

Note: Ensure that B-ME is added to Buffer RLT before use.



Table 1. Buffer RLT volumes for RNeasy Midi isolation of total RNA from animal

tissues

RNeasy column Amount of tissue (mg) Buffer RLT (ml)

Midi 20–75 2.0

Midi 75–130 2.0 (4.0)

Midi 130–250 4.0



2) Centrifuge the tissue lysate for 10 min at 3000–5000 x g. Carefully transfer the supernatant to a new 10–15 ml tube by pipetting. Use only this supernatant (lysate) in subsequent steps.

3) Add 1 volume (2.0 ml or 4.0 ml) of 70% ethanol to the homogenized lysate, and mix immediately by shaking vigorously. Ensure that any precipitates are resuspended. Do not centrifuge. Continue without delay with step 4.

4) Apply the sample to an RNeasy midi column placed in a 15 ml centrifuge tube and close the tube gently. Maximum loading volume is 4.0 ml. Centrifuge for 5 min at 3000–5000 x g. Discard the flow-through.

If the volume exceeds 4.0 ml, load aliquots successively onto the RNeasy column, and centrifuge as above. Discard the flow-through after each centrifugation step.

5) Pipet 2.0 Buffer RW1 into the RNeasy column. Centrifuge for 5 min at 3000–5000 x g to wash. Discard the flow-through.

6) Perform an on-column DNase digestion by adding 20 µl DNase I stock solution (dissolve the solid DNase I (1500 Kunitz units) in 550 µl of the RNase-free water. Mix gently by inverting the tube. Do not vortex.) to 140 µl Buffer RDD and mix by gently flicking the tube. Centrifuge the tube briefly to collect residual liquid from the sides of the tube. Pipet the DNase I incubation mix directly onto the RNeasy silica-gel membrane and place on the benchtop (20–30°C) for 15 min.

7) Pipet 2.0 ml Buffer RW1 into the RNeasy column, and place on the

benchtop for 5 min. Then centrifuge for 5 min at 3000–5000 x g. Discard the flowthrough.

8) Add 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 2 min at 3000–5000 x g to wash the column. Discard the flow-through.

9) Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 5 min at 3000–5000 x g to dry the RNeasy silica-gel membrane.

10) To elute, transfer the RNeasy column to a new 15 ml collection tube. Pipet the appropriate volume of RNase-free water (see Table 2) directly onto the RNeasy silica-gel membrane. Close the tube gently. Let it stand for 1 min, and then centrifuge for 3 min at 3000–5000 x g.

Table 2. RNase-free water volumes for RNeasy Midi elution RNeasy column

Expected total RNA yield RNase-free water

Midi ≤150 µg 150 µl

Midi 150 µg – 1 mg 250 µl

11) Repeat the elution step (step 10) as described with a second volume of RNase-free water. To obtain a higher total RNA concentration, this second elution step may be performed using the first eluate (from step 11). The yield will be 15–30% less than the yield obtained using a second volume of RNase-free water, but the final concentration will be higher.
(Parameters: Extracted product = total_RNA, Amplification = none) At 8.5 days of development the dam was culled by cervical dislocation, the uterine horns disscted and placed into PBS. The uterus was dissected to release decuduae and these opened according to standard protocols. Morphologically normal looking embryos of between 4 and 6 somites were collected. The left and right lateral plates were dissected away with watchmakers forceps and pools of 4 left and 4 right lateral plates were snap frozen in LN2. C3H/HeH females were mated with 101/H males

Mating was assessed by the presence of vaginal plugs the next morning. Identification of plug was designated day 0.5.



At 8.5 days of development the dam was culled by cervical dislocation, the uterine horns disscted and placed into PBS. The uterus was dissected to release decuduae and these opened according to standard protocols. Morphologically normal looking embryos of between 4 and 6 somites were collected. The left and right lateral plates were dissected away with watchmakers forceps and pools of 4 left and 4 right lateral plates were snap frozen in LN2.
(Parameters: start time = 8.5, time unit = days, temperature unit = C) Equal amounts of RNA were pooled from each sample SMART cDNA labelling protocol – Cy3



1st Strand cDNA Synthesis



1.For each sample, combine the following reagents in a sterile, thin wall 0.2 ml tube:

1 µg total RNA

1µl 3’ SMART CDS Primer IIA (10 µM)

(5’-AAGCAGTGGTATCAACGCAGAGTAC-T30VN-3’)

1µl SMART IIA Oligonucleotide (10 µM)

(5’-AAGCAGTGGTATCAACGCAGAGTACGC)r(GGG)-3’)

xµl dH2O

5µl Total volume

2.Mix contents and spin the tube briefly in a microfuge.

3.Incubate at 72°C for 2 min (in Tetrad, use heated lid).

4.Cool the tube on ice for 2 min.

5.Spin tube briefly to collect contents.

6.Add the following to each tube:

2µl 5x First-strand buffer

1µl DTT (20 mM)

1µl 50x dNTP (10 mM)

1µl PowerScript RT (Clontech)

7.Mix by gentle pipetting and spin tubes briefly in microfuge.

8.Incubate the tubes at 42°C for 1 hr (in Tetrad, use heated lid).

9.Dilute the first-strand reaction product with 40ul TE buffer.

10.Heat the tubes at 72C for 7 mins.

11.Place tubes on ice to terminate first-strand synthesis.



cDNA Amplification by LD PCR



1.Transfer a 2µl aliquot from the first-strand reaction to a clean, prechilled 0.2 ml tube, place on ice. Store any unused first-strand reaction mixture at –20C.

2.Preheat Tetrad to 95°C.

3.Prepare a master mix for all reaction tubes, plus one additional tube. Combine the following components in the order shown:

Per Rn

75µl dH2O

10µl 10x PCR buffer II

5µl 50mM MgCl2

2µl 50x dNTP (10 mM)

4µl 5’ PCR Primer IIA (10 µM)

(5’-AAGCAGTGGTATCAACGCAGAGT-3’)

2µl Amplitaq polymerase (Clontech)

98µl Total Volume

4.Mix by vortexing and spin tube briefly in microfuge.

5.Aliquot 98µl of Master Mix into each reaction tube.

6.Mix contents by gently flicking tube, spin briefly in microfuge to collect contents.

7.Place tube into the preheated thermocycler, commence thermocycling using the following programme:

95°C 1 min

X cycles: (X depends on amount of input RNA, for 1 g 16 cycles is sufficient)

95°C 5 sec

65°C 5 sec

68°C 6 min

8.Store ds cDNAproduct at -20C prior to labelling



Klenow labelling



1.Transfer a 21µl aliquot of ds cDNA to a fresh 0.2µl thin wall tube

2.Add 20µl 2.5x Random Primer Reaction Buffer (Invitrogen)

3.Incubate at 95°C for 5 min, then place on ice

4.Add:

5µl 10x low-C dNTP mix (1.2 mM each dATP, dGTP, and dTTP

0.6 mM dCTP)

3µl Cy3 dCTP

1µl Klenow

5.Incubate 37°C for 3h (Tetrad)

6.Stop reaction by adding 5µl stop buffer (Invitrogen)



Purification of labelled probes



1.Resuspend the resin in a G-50 column by vortexing gently

2.Snap off bottom closure, loosen cap a quarter turn and place column in a 1.5 ml microfuge tube

3.Pre-spin column 2000g for 1 min to remove buffer. Blot tip of column dry on a clean paper towel

4.Remove top cap and place tube in a fresh 1.5ml tube.

5.Pipette the sample onto the centre of the angled surface of the resin bed being careful not to disturb the resin (purify Cy3 and Cy5 separately)

6.Centrifuge 2000g for 1 min, discard the column.




(Parameters: Mass unit = Micro gram, Label used = Cy3, Amplification = PCR) SMART cDNA labelling protocol – Cy5



1st Strand cDNA Synthesis



1.For each sample, combine the following reagents in a sterile, thin wall 0.2 ml tube:

1 µg total RNA

1µl 3’ SMART CDS Primer IIA (10 µM)

(5’-AAGCAGTGGTATCAACGCAGAGTAC-T30VN-3’)

1µl SMART IIA Oligonucleotide (10 µM)

(5’-AAGCAGTGGTATCAACGCAGAGTACGC)r(GGG)-3’)

xµl dH2O

5µl Total volume

2.Mix contents and spin the tube briefly in a microfuge.

3.Incubate at 72°C for 2 min (in Tetrad, use heated lid).

4.Cool the tube on ice for 2 min.

5.Spin tube briefly to collect contents.

6.Add the following to each tube:

2µl 5x First-strand buffer

1µl DTT (20 mM)

1µl 50x dNTP (10 mM)

1µl PowerScript RT (Clontech)

7.Mix by gentle pipetting and spin tubes briefly in microfuge.

8.Incubate the tubes at 42°C for 1 hr (in Tetrad, use heated lid).

9.Dilute the first-strand reaction product with 40ul TE buffer.

10.Heat the tubes at 72C for 7 mins.

11.Place tubes on ice to terminate first-strand synthesis.



cDNA Amplification by LD PCR



1.Transfer a 2µl aliquot from the first-strand reaction to a clean, prechilled 0.2 ml tube, place on ice. Store any unused first-strand reaction mixture at –20C.

2.Preheat Tetrad to 95°C.

3.Prepare a master mix for all reaction tubes, plus one additional tube. Combine the following components in the order shown:

Per Rn

75µl dH2O

10µl 10x PCR buffer II

5µl 50mM MgCl2

2µl 50x dNTP (10 mM)

4µl 5’ PCR Primer IIA (10 µM)

(5’-AAGCAGTGGTATCAACGCAGAGT-3’)

2µl Amplitaq polymerase (Clontech)

98µl Total Volume

4.Mix by vortexing and spin tube briefly in microfuge.

5.Aliquot 98µl of Master Mix into each reaction tube.

6.Mix contents by gently flicking tube, spin briefly in microfuge to collect contents.

7.Place tube into the preheated thermocycler, commence thermocycling using the following programme:

95°C 1 min

X cycles: (X depends on amount of input RNA, for 1 µg 16 cycles is sufficient)

95°C 5 sec

65°C 5 sec

68°C 6 min

8.Store ds cDNAproduct at -20C prior to labelling



Klenow labelling



1.Transfer a 21µl aliquot of ds cDNA to a fresh 0.2µl thin wall tube

2.Add 20µl 2.5x Random Primer Reaction Buffer (Invitrogen)

3.Incubate at 95°C for 5 min, then place on ice

4.Add:

5µl 10x low-C dNTP mix (1.2 mM each dATP, dGTP, and dTTP

0.6 mM dCTP)

3µl Cy5 dCTP

1µl Klenow

5.Incubate 37°C for 3h (Tetrad)

6.Stop reaction by adding 5µl stop buffer (Invitrogen)



Purification of labelled probes



1.Resuspend the resin in a G-50 column by vortexing gently

2.Snap off bottom closure, loosen cap a quarter turn and place column in a 1.5 ml microfuge tube

3.Pre-spin column 2000g for 1 min to remove buffer. Blot tip of column dry on a clean paper towel

4.Remove top cap and place tube in a fresh 1.5ml tube.

5.Pipette the sample onto the centre of the angled surface of the resin bed being careful not to disturb the resin (purify Cy3 and Cy5 separately)

6.Centrifuge 2000g for 1 min, discard the column.
(Parameters: Label used = Cy5, Amplification = PCR, Mass unit = Micro gram) The labels were re-suspended in 40?l of hybridisation buffer (40% deionised formamide; 5x Denhardt’s solution; 5x SSC; 1mM sodium pyrophosphate; 50mM Tris-HCl pH 7.4; 0.1% SDS) and hybridised to the arrays overnight at 48oC in a water bath using Corning Hybridisation Chambers. After hybridisation, the arrays were then washed initially in 500ml of 2xSSC, then for 5 minutes with vigorous shaking in 500ml 0.1x SSC, 0.1% SDS and then finally in 500ml 0.1xSSC for 2 minutes with vigorous shaking.
(Parameters: Chamber type = Corning Microarray Technology- CMT-Hyb chamber, Quantity of label target used = 500, Mass unit = Nano gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 48) Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology SDRF File E-MEXP-2277.sdrf.txt Term Source Name EFO NCBI Taxonomy The MGED Ontology NCI_thesaurus ArrayExpress The MGED Ontology EFO Term Source File http://www.ebi.ac.uk/efo/ http://www.ncbi.nlm.nih.gov/Taxonomy/ http://mged.sourceforge.net/ontologies/MGEDontology.php ncithesaurus.obo.alt http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version