Investigation Title Hodgkin_IV-IIIB_Ben-Dov_CRG
Comment[AdditionalFile:XLSX] Ben-Dov_C_HD.xlsx
Comment[Submitted Name] Hodgkin_IV-IIIB_Ben-Dov_CRG
Experimental Design cell_type_comparison_design co-expression_design all_pairs transcription profiling by array
Experimental Design Term Source REF The MGED Ontology The MGED Ontology EFO
Comment[AEMIAMESCORE] 5
Comment[SecondaryAccession]
Comment[ArrayExpressReleaseDate] 2010-12-31
Comment[ArrayExpressAccession] E-MEXP-2959
Comment[MAGETAB TimeStamp_Version] 2010-10-29 15:55:29 Last Changed Rev: 14857
Experimental Factor Name CELL_TYPE
Experimental Factor Type cell_type
Experimental Factor Term Source REF
Person Last Name Ben-Dov
Person First Name Claudia
Person Mid Initials P
Person Email claudia.bendov@crg.es
Person Phone +34 933160100
Person Fax
Person Address Gene Regulation, Dr. Aiguader 88, Barcelona, Barcelona, 08003, Spain
Person Affiliation Center for Genomic Regulation
Person Roles submitter
Person Roles Term Source REF The MGED Ontology
Quality Control Type biological_replicate dye_swap_quality_control
Quality Control Term Source REF The MGED Ontology The MGED Ontology
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2010-12-31
PubMed ID
Publication DOI
Publication Author List
Publication Title
Publication Status
Publication Status Term Source REF
Experiment Description Hodgkin lymphoma derived cell lines (from the Deutsche Sammlung von Mikroorganismen und Zellkulturen) were cultured in RPMI 1640 medium supplemented with
20% (HDML-2) or 10% (HD-MY-Z) heat inactivated fetal calf serum. Cytoplasmic RNA was extracted according to "Cytoplasmic_RNA_CRG" protocol available at Arrayexpress
Protocol Name P-MTAB-17626 P-MTAB-17627 P-MTAB-17629 P-MTAB-17628
Protocol Type nucleic_acid_extraction labeling hybridization bioassay_data_transformation
Protocol Description 1) Cytoplasmic RNA, including on column DNAse treatment, was extracted from Hodgkin cell lines with RNeasy® Mini Kit from QIAGEN according to manufacturer's instructions.
http://www.qiagen.com/selectlocation.aspx?redirect=%2fliterature%2frender.aspx%3fid%3d576.
2) Samples were quantified by NanoDrop and subject to quality control with Bioanalyzer (Agilent).
(Parameters: Extracted product = OTHER: cytoplasmic RNA, Amplification = none) Agilent Low RNA Input Fluorescent Linear Amplification Kit (5184-3526)
NOTE: in some steps there are two different amounts X/Y. "X" is according to the original protocol."Y" is according to a modified protocol (Syed HA, Threadgill DW. Enhanced oligonucleotide microarray labeling and hybridization. Biotechniques. 2006 Dec;41(6):685-6).
A) cDNA Synthesis
0. Pre warm the 5X first strand buffer 3-4 at 65°C, vortex, spin and keep it at RT
1. Add to a 1.5 ml Eppendorf tube 500 ng total RNA in 9.3/ 5.8 ?l H2O (Ambion)
2. Add 1.2/ 0.8 ?l of Oligo dT-T7 promoter primer (Agilent Kit) + 1/ 0.67 ?l random Nanomer-T7 (CGTAATACGACTCACTATAGGGNNNNNNNNN; HPLC purified)
3. Incubate at 65°C for 10 (block)
4. Ice 5
5. Prepare the following Master mix
1X 1X
5X Buffer 4 ?l / 2 ?l
0.1M DTT 2 ?l / 1 ?l
10mM dNTPmix 1 ?l / 0.5 ?l
MMLV RT 1 ?l / 0.6 ?l
RNAse out 0.5 ?l/ 0.3 ?l
TOTAL 8.5 ?l/ 4.4 ?l
6. Add 8.5/4.4 ?l master mix to the tube (step 4).
7. Incubate at 40°C 2 hs (in water bath), cover the tubes with parafilm.
8. Incubate 65°C 15 (block).
9. Ice 5 and spin.
B. cRNA SYNTHESIS
NOTE: From now avoid exposure to light
1. Add 2.4/ 0.5 ?l Cy3 or Cy5-CTP 1mM to the former tube
2. Prepare the following Master mix
Pre warm the 50% PEG 1 at 40°C in water bath, vortex and spin keep at RT
1X 1X
H2O 15.3 ?l /3.83 ?l
4X Transcription buffer 20 ?l /5 ?l
0.1M DTT 6 ?l /1.5 ?l
NTPmix 8 ?l /2 ?l
50% PEG 6.4 ?l /1.6 ?l
RNAse out 0.5 ?l /0.12 ?l
Inorganic pyrophosphatase0.6 ?l /0.15 ?l
T7 RNA polymerase 0.8 ?l /0.3 ?l
TOTAL 57.6 ?l /14.50 ?l
3. Add 57.6/14.5 ?l mix to the tube, soft vortex and spin.
4. Incubate at 40°C 2 hs in water bath. Cover the tubes with parafilm.
5. Cover the water bath to avoid light exposure
C. cRNA PURIFICATION (Qiagen RNAeasy mini kit)
1. Add 20/ 80 ?l H2O to former tube (to reach 100 ?l)
2. Add 350 ?l buffer RLT, short vortex
3. Add 250 ?l 100% Ethanol, mix well by pipetting.
4. Transfer sample to column, spin 30, 13000 rpm, 4°C
5. Transfer column to new tube and add 500 ?l buffer RPE, spin 30 13000 rpm, 4°C
6. Repeat, add 500 ?l buffer RPE, spin 3, 13000 rpm, 4°C
7. Add 30 ?l H20, wait 1, spin 30, 13000 rpm, 4°C
8. Repeat, Add 30 ?l H20, wait 1, spin 30, 13000 rpm, 4°C
9. Aliquot 3?l for Bioanalyzer and NanoDrop
10. Store rest of sample at -80°C
Expected yield: 400ng/??l; 30-40 pmol dye/ ?g.
120-170ng/??l; 30-40 pmol dye/ ?g
(Parameters: Mass unit = Micro gram, Amplification = none) FRAGMENTATION AND HYBRIDIZATION.
Agilent In situ hybridization kit plus (5184-3568)
1. Prepare 5.5-6.75 ug of each labeled-cRNA in the same tube. Equal amount of both samples, and similar pmol dye/ug of cRNA according to the NanoDrop. (Up to 3-4 units of difference)
2. Add 50 ul of labeled controls from Agilent.
3. Add 10 ul of 25X fragmentation buffer.
4. Add H2O up to 250 ul.
5. Incubate 30 at 60ºC. Cover the tubes with parafilm.
6. Cover the water bath with aluminum to avoid light exposure.
7. Remove the tube and add 250 ul of hybridization buffer. Mix by pippetting avoiding bubbles formation.
8. Set up the hybridization chamber (Agilent, Cat.#: G2534A) and add the solution on top of the glass slide.
9. Hybridize 60ºC 18hs. at 6 RPM.
WASHING OF THE ARRAY
- 20x SSC is commercial from AMRESCO
- Triton is coming with Agilent hybridization kit
Wash solution 1.
MiliQ water (steril) 350 ml
20X SSC 150 ml
10% Triton X-102 250 ?l
TOTAL 500 ml
Wash solution 2.
MiliQ water (steril) 498 ml
20X SSC 2.5 ml
10% Triton X-102 250 ?l
TOTAL 500 ml
FILTER both solutions with Stericup from Milipore. Keep solution 1 at RT, and 2 at 4ºC.
- Both solutions should be fresh
- It is critical to perform the whole process in an atmosphere free of ozone and light.
1. Remove the array from the chamber inside a can with wash solution 1, try to avoid contact with air.
2. Quickly, put the array inside another box (black) containing wash solution 1, and cover the box. Shake it for 10 at RT at 150 RPM.
3. Pass the slide to a new box containing cold wash solution 2. Shake it inside an ice box for 5 at 150 RPM.
4. Dry the slide with Nitrogen pistol.
5. Assembly the scanner chamber.
(Parameters: Chamber type = OTHER: Agilent, Cat.#: G2534A, Quantity of label target used = 6, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume unit = Micro litre, temperature = 60) The background correction method used in the analysis was Normexp (Ritchie, et al., 2007). Locally weighted linear regression (LOWESS) analysis was used as a normalization method (Smyth G.K.et al., 2003)
-Smyth, G. K., and Speed, T. P. (2003). Normalization of cDNA microarray data. Methods 31, 265-273.
-Ritchie, ME, Silver, J, Oshlack, A, Holmes, M, Diyagama, D, Holloway, A, and Smyth, GK (2007). A comparison of background correction methods for two-colour microarrays. Bioinformatics 23, 2700-2707
Protocol Parameters
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology
SDRF File E-MEXP-2959.sdrf.txt
Term Source Name The MGED Ontology EFO ArrayExpress The MGED Ontology EFO
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/
Term Source Version