nucleic acid labeling protocol
Hybridization of Oligo Microarrays with ULS-CY Dye labeled amplified RNA, using the TECAN HS4800 Hybridization Station. This protocol can be used to label amplified antisense RNA with the ULS system from KREATECH Biotechnology using the Amersham Cy Dyes (kit # EA-006). Make sure that the amplified RNA is as pure as possible! If Qiagen RNeasy columns are used in final purification step in amplification, make sure that no salts are left on the membrane of the column. This can be prevented by washing the RNA bound to the column twice with 80% ethanol, before eluting the aRNA. 1) For every reaction, pipet 2 ul 10X labeling solution into a 200 ul PCR tube. 2) Add 1 ug aRNA in a maximal volume of 17 ul. 3) Add 0.3 ul of ULS-Cy5 or 1 ul of ULS-Cy3 label. 4) Adjust the total volume to 20 ul with water and mix the solution by pipetting. 5) Incubate at 85 degrees C for 30 minutes. 6) Let the mixture cool to room temperature for about three minutes. 7) Shake a KREApure column (use a separate column for each reaction) to make sure that the column material is completely mixed (shake the material down before unscrewing the lid). 8) Break of the bottom part, remove the lid and place the column in a 2 ml Eppendorf vial without lid. 9) Spin the column for 1 minute at maximum speed. 10) Load the labeling mixture onto the column and place it in a clean 2 ml Eppendorf vial without lid, but keep the lid to close the vial after purification (make sure that you load the mixture on the highest part of the material: The fixed-angle rotor of the centrifuge forces the column material to one side of the column). 11) Place the highest part of the column material on the outside of the rotor and spin for 1 minute at maximum speed. 12) Discard the column and keep the eluate (about 20 ul). Analysis of labeled aRNA 13) Measure the amount of purified and labeled aRNA, and frequency of Dye incorporation (FOI) on the NanoDrop spectrophotometer with 2 ul of each labeled aRNA. FOI (number of Dye molecules per kb of aRNA) = (324.5 * pmol dye/ul) / RNA ng/ul 14) Pool the two purified reactions (total volume approximately 40 ul) and SpeedVac the mixture until a volume of 9 ul is reached. 15) Transfer the mixture to a 200 ul PCR tube and add 1 ul Fragmentation buffer to decrease the fragment size to 60-200 bases. 16) Incubate at 70 degrees C for 15 minutes. 17) Spin the vial briefly and add 1 ul of stop solution, mix by pipetting (the labeled aRNA can form aggregates which dissolve by pipetting) and place on ice until further use. 18) Transfer the mixture to a 1.5 ml Eppendorf vial and add 6 ul of blocking solution (containing 20 ug Poly d(A), 8 ug yeast t-RNA and 20 ug COT-1 DNA). 19) Add 43 ul of RNAse-free water and store at 42 degrees C. Note: If the labeled material is not used the same day for hybridization, it can be stored at -20 degrees C or -80 degrees C until further use. Samples can be stored for several weeks before use. If you are planning to do a large series of hybridizations in a short period of time, we recommend to first label all your aRNA before starting with the hybridizations. Used materials: ULS-Cy5* EA-006 KREATECH Biotechnology ULS-Cy3* EA-006 KREATECH Biotechnology KREApure column* EA-006 KREATECH Biotechnology 10x labeling Solution* EA-006 KREATECH Biotechnology *part of the kit RNA Fragmentation Reagents 8740 Ambion COT-1 DNA 15279-011 Invitrogen Poly d(A) 27-7836-01 Pharmacia Yeast t-RNA 109 495 Roche
Data from multiple normalizations are combined using a weighted mean where the weight is proportional to the predicted error by the errormodel.
normalization data transformation protocol
Raw data is smoothed using a lowess fit per subarray.
nucleic acid hybridization to array protocol
Hybridization of microarrays in a TECAN Hs4800 Hybridization Station 1) Store labeled probe at 42 degrees C until use 2) Make fresh 2X F-hybridization buffer (500 ul 100% formamide + 500 ul 20X SSC + 10 ul 20% SDS). Heat the buffer to 42 degrees C for at least five minutes and add 60 ul to the labeled probe (final volume should be 120 ul), mix the 2X F-hybridization buffer well before adding, store probe at 42 degrees C until use. 3) Thaw a vial of pre-hybridization buffer and keep at 42 degrees C until use. Note: Make a pre-hybridization buffer stock which contains 5X SSC, 0.1% SDS and 1% BSA and aliquot into 2 ml vials. First make a filtered 10% BSA stock solution which can be added to the buffer. Store the pre-hybridization buffer at -20 degrees C and thaw only once. 4) Place microarray in the hybridization station and start the program as mentioned below. 5) Add the pre-warmed pre-hybridization buffer (mix well before use) to the microarray at Step 2 of the TECAN protocol. 6) When the hybridization station has reached Step 6, heat the labeled probe to 95 degrees C for three minutes in a dry heat block. Quickly spin the vial to collect the labeled probe in the bottom and pipet it onto the microarray. TECAN HS4800 Hybridization Station Protocol Step 1 : Wash 42.0C, Repeats: 1, Solution - 5X SSC 0,1 %SDS, Time: 0:00:30, Soak time: 0:00:30 Step 2 : Probe Injection 42.0C Step 3 : Hybridization 42.0C, Agitation Frequency: Medium, Time:1:00:00 Step 4 : Wash 42.0C, Repeats: 2, Solution - MilliQ Water, Time: 0:02:00, Soak time: 0:01:00 Step 5 : Wash 42.0C, Repeats: 1, Solution - 5X SSC 0,1 %SDS, Time: 0:00:55, Soak time: 0:00:00 Step 6 : Probe Injection 42.0C Step 7 : Hybridization 42.0C, Agitation Frequency: Medium, Time: 16:00:00 Step 8 : Wash 42.0C, Repeats: 1, Solution - 5X SSC 0,1 %SDS, Time: 0:01:00, Soak time: 0:01:00 Step 9 : Wash 42.0C, Repeats: 2, Solution - 2X SSC 0,1 %SDS, Time: 0:01:00, Soak time: 0:01:00 Step 10 :Wash 42.0C, Repeats: 2, Solution - 1X SSC, Time: 0:01:00, Soak time: 0:01:00 Step 11 :Wash 23.0C:,Repeats: 2, Solution - 0.2X SSC, Time: 0:00:45, Soak time: 0:00:45 Step 12 :Slide Drying 30.0C: Nitrogen - Time: 0:02:00 Used materials: 20 x SSC #51232 BioWhittaker 20% SDS solution #19812323 BioSolve BV Formamide #F 7503-1000 Sigma-Aldrich Chemie BV Bovine Serum Albumin, Fraction V #A 9647-500g Sigma-Aldrich Chemie BV
array scanning and feature extraction protocol
ImaGene 6.0 Quantification of Oligo Microarrays Raw tiff images are first splitted, rotated and flipped along the opposite diagonal using the Agilent TiffSplitter (version #A.6.1.1) The two newly created tiff images (Red and Green) are opened in ImaGene (Biodiscovery, version #6.0.1), with the followng settings: Spot Finding Find Negative Spots on Enforce Grid Constraints on (Local Flexibility 5.0 pixels) (Global Flexibility 50%) Segmentation Auto Segmentation on Quality Flags Empty Spots on (Threshold 1.0) Poor Spots on Negative spots on Measurements Mean on Median on Total on Standard Deviation on Area on Spot Area on Shape Regularity on A GeneID which matches the specific microarray is loaded, followed by loading a grid to fit the spots on the array, the grid settings as below: Metarows 12 Metacolumns 4 Rows 28 Columns 28 Minimal Diameter 8.0 Maximal Diameter 18.0 The 'Auto Adjust Grid' function is used to correctly place the grid over the spots. A visual check is performed to make sure the grid is correctly placed, before using the 'Auto Adjust Spots' to determine the borders of the spots. The final step is to Quantify ('Make Measurements' function) the spots before the data can be saved.
nucleic acid extraction protocol
RNA was isolated using the RNeasy Micro kit (Qiagen, Hilden, Germany). Isolated total RNA was subsequently DNase-treated by using the Qiagen RNase-free DNase kit (Qiagen, Hilden, Germany) and dissolved in RNase-free H2O. cDNA was generated by Superscript II reverse transcriptase using total RNA as template and an oligo(dT) primer containing a T7 polymerase recognition site. Amplified RNA was generated by in vitro transcription using T7 RNA polymerase (Megascript T7 kit, Ambion, Austin, TX). Amplification yields were 1000- to 2000-fold. The same amount of amplified RNA was used for each sample.
Wild type (WT) or Cd27-/- OT-I mice of 8-12 weeks old were immunized intranasally with 500 ug ovalbumin (OVA) protein and 1 ug cholera toxin in 50 ul HBSS. Three, four or eight days (as indicated) after immunization, OT-I T cells were isolated from the mediastinal draining lymph node (DLN), the spleen or the lung using flow cytometric cell sorting on a FACSAria (BD). Around 1 million cells of each sample were obtained. These cells were spun down and the pellet was frozen in liquid nitrogen and kept at -80C before RNA extraction.