title: Baylor/AZ_Mouse_Cytokine_Array
status: Public on Aug 23 2007
contact.city: Macclesfield
contact.first: stephanie
contact.middle: ann
contact.last: roberts
contact.email: stephanie.roberts@astrazeneca.com
contact.state: Cheshire
contact.address: Alderley Park
contact.department: RIRA
contact.country: United Kingdom
contact.institute: astrazeneca
organism: Mus musculus
description: R & D Systems mouse cytokine array represents a comprehensive collection of cytokines, chemokines and other immunomodulatory factors as well as the receptors for these categories of molecules. The array consists of 514 different cloned cDNAs, printed as PCR products, on a positively charged nylon membrane. The amplified cDNA sequences contain coding sequence, avoiding repetitive sequence elements that are often associated with untranslated regions. All the cDNA sequences are verified prior to PCR. Each cDNA is printed in duplicate with 10ng of DNA per spot. In addition, eight positive control "housekeeping" genes, six negative controls and mouse genomic DNA are included on the array.
manufacture_protocol: The manufacturers recommend the purification of total RNA using Qiagen RNeasy Maxi Kit. If the mouse Cytokine-specific Primers are being used then isolation of poly (A)+ RNA is not necessary.
  The protocol recommends that the quality of RNA is checked using a spectrophotometer and agarose gel electrophoresis, the RNA sample should only be used if it is not degraded and there is no evidence of genomic contamination. In this experiment total RNA was isolated from the mouse joint samples and quantified and quality checked pre and post DNAse treatment using Agilent Technologies 2100 Bioanalyzer.
  Radiolabelled cDNA is generated using Mouse Cytokine-specific primers. The cDNA labelling reaction involves two steps. First, the gene-specific primers are annealed to the RNA sample. Second, a radiolabelled nucleotide, dNTPs, buffer, RNase inhibitor and reverse transcriptase are added to promote cDNA synthesis. For radiolabelling R&D Systems recommends the use of 33P rather than 32P. The protocol recommends the use of 2ug of total RNA or 0.5ug of poly (A)+ RNA for the reaction. In this experiment it was found that 5ug Total RNA was optimal for the reaction.
  The annealing reaction is assembled in a 0.5ml tube with; 4ul Mouse Cytokine specific primers + Total RNA, the volume is adjusted to 15ul using RNAse-free water. The annealing reaction is carried out by placing the reaction in a thermal cycler. The cycle used is 90oC for 2 mins, then ramp to 42oC over a period of 20 mins. Once 42oC is reached the reaction is centrifuged and the following components were added; reverse transcriptase buffer, dNTPs, alpha33P dCTP, RNAse inhibitor, AMV reverse transcriptase, RNAse free water. The final reaction volume is 30ul. The reaction is mixed and returned to the thermal cycler and incubated at 42oC for 2-3 hrs.
  The labelled cDNA is then purified. Unincorporated nucletides need to be removed to minimize non-specific background hybridizaton to the arrays. A Sephadex G-25 spin column is used to do this. The sample is placed down the column and centrifuged at 110 x g for 4 mins. the eluate is saved as it contains the purified cDNA sample.
  The labelling is assessed before and after column purification. The % incorporation is monitored using a scintillation counter. Typically incorporation shoudl be between 20-50%.
  Hybridization:
  1.Arrays are rinsed in 50ml 2X SSPE at room temp for 5 mins.the arrays are drained.
  2. Denature Salmon Testes DNA in boiling water bath for 5 mins. Place on ice. Add to the hybridization solution before use to a final concentration of 100ug/ml.
  3.pre-warm teh hybridization oven and the hybridization solution from step 2 to 65oC. Pre-Hybridize the cDNA array in 5ml hybridization solution for at least 1 hr at 65oC, in a roller bottle rotating at 6 rpm.
  4. add all the labelled cDNA t 2-3 ml hybridization solution in a 15ml screw-top conical tube. Denature the cDNA at 90-95OC for 10 mins in a water bath.
  5. Decant and discard the Hyb solution from the pre-hybridized array. Add the denatured labeled cDNA in teh Hyb solution to the array in the roller bottle
  6. Hybridized overnight (12-18hrs) at 65oC
  7. Decant the Hyb solution
  8. Add 40-50ml of wash solution1 to the roller bottle (0.5 x SSPE, 1% SDS). Wash the array at room temp for 3 mins. Discard the wash solution.
  9. Repeat step 8 twice.
  10. Pre-warm the wash solution to 65oC. Add 80-100ml Wash solution 1. Wash arrays in the hybdridization oven at 65oC for 20 mins (6rpm). Discard wash solution.
  11.Repeat step 10 once.
  12. Pre-Warm the wash solution II to 65oC (0.1 x SSPE, 1% SDS). Add 80-100ml wash solution II to the roller bottle. Wash arrays at 65oC for 20 mins (6rpm). Discard the wash solution.
  13. Remove the array from the roller bottle. Lay the array on a sheet of blotting paper.
  14. Air-Dry the array for 5 mins.
  15. Wrap the array in clear plastic wrap and subject it to autoradiography. In this experiment arays were scanned using a phosphoimager. The protocol recommends scanning images at 50um. Typically an overnight exposure to a phosphoimager screen should be sufficient.
  Quantitation of gene expression signals is best determined from phosphoimager generated image files. The image files may be analysed using the phophoimager manufacturers software eg in this experiment ArrayVision software from Imaging Research, Inc. was used.