CD38- sub-clones derived from a CLL patient with bimodal expression of the CD38 antigen
Extracted molecule
total RNA
Extraction protocol
Trizol reagent was added the cell-sorted populations (1 x 107 cells), and this was placed in tubes containing Phase Lock Gel-Heavy and were incubated for 5 minutes at 37oC. 0.2ml of chloroform was added and the tubes were shaken. The samples were spun at 12,000 x g for 10 minutes at 2-8oC, to separate the phases (the clear aqueous phase was on top and cloudy chloroform phase was on the bottom). The aqueous phase was transferred to a clean tube, and to which 0.6ml of isopropyl alcohol was added. The tubes were shaken, and spun as before. The pellet of RNA was re-suspended in 75% ethanol and centrifuged at 7,500 x g for 5 minutes at 2-8oC. The supernatant was removed, and the pellet allowed to air dry. The pellet was re-suspended in RNase-free water, and incubated at 65oC for at least 30 minutes. The RNA was quantified using the Nanodrop 1000 (Nanodrop Technologies), and the purity was checked by running an Agilent chip on the Agilent bioanalyzer (Agilent Technologies). One-cycle cDNA synthesis A 1:20 dilution of Poly-A (poly-adenine) control stock was made in Poly-A Control Dilution Buffer from the commercially available kit (Affymetrix). From this solution, 2ul was added to Poly-A control dilution buffer to make a 1:50 dilution. Again, 2ul was taken from this solution and added to Poly-A control dilution buffer to make a 1:10 dilution. 2ul of this final dilution was added to the sample of RNA. T7-Oligo(dT) Primer was added to the RNA and the volume was made up to 12ul with RNase-free water. The reaction was incubated at 70oC for 10 minutes and then cooled for at least 2 minutes. The First-Strand master mix was made up as follows: 4ul of 5X 1st strand master mix, 2ul 0.1M Dithiothreitol (DTT) and 1ul 10mM dNTP. This was added to the RNA/T7-oligo(dT) primer mix and incubated at 42oC for 2 minutes. The appropriate amount of SuperScript II was added to each RNA sample and incubated for 1 hour at 42oC. The sample was then cooled at 4oC for at least 2 minutes. The Second-Strand master mix was made up as follows: 91ul of RNase-free water, 30ul 5X 2nd strand reaction mix, 3ul 10mM dNTP, 1ul E. coli DNA ligase, 4ul of E.coli DNA polymerase I and 1ul RNase H. 130ul of this master mix was added to each RNA sample and it was incubated for 2 hours at 16oC. T4 DNA Polymerase (2ul) was added to the sample and was incubated at 16oC for 2 minutes. 10ul of 0.5M ethylene diamine tetra-acetic acid (EDTA) was added to the sample. cDNA (compliment DNA) Binding Buffer (600ul) was added to the sample making the mixture yellow. The sample was applied to the cDNA Cleanup Spin Column and spun at ≥ 8,000 x g for 1 minute. The spin column was transferred into a new collection tube and cDNA Wash Buffer was added to the column. This was then spun at ≥ 8,000 x g for 1 minute and the flow through was discarded. The caps of the spin columns were opened, centrifuged for 5 minutes at maximum speed and the collection tube and flow-through were discarded. The column membranes were dried by centrifugation and the spin columns were placed in clean collection tubes. cDNA elution buffer was added directly onto the column membrane and incubated for 1 minute at room temperature. This was then spun at maximum speed for 1 minute to elute.
Label
biotin
Label protocol
The following solution was made up; 4ul 10X IVT labelling buffer, 12ul IVT (in vitro transcription) labelling NTP (nucleotide triphosphate) mix, 4ul IVT labelling enzyme mix, required amount of template cDNA and the volume made up to 40ul with RNase-free water. The mixture was incubated at 37oC overnight. The following day 60ul of RNase-free water, 350ul cRNA binding buffer and 250ul ethanol was added to the sample, which was applied to the IVT cRNA Cleanup Spin Column. This was spun at >8,000 x g for 15 seconds, and the flow through and collection tube was discarded. The spin column was placed in a new collection tube. 0.5ml IVT cRNA wash buffer was added to the column and was spun as before with the flow through being discarded. 80% ethanol (0.5ml) was added to the sample and was spun as before. The spin column’s cap was opened and it was spun at maximum speed for 5 minutes. The flow through and collection tube was discarded. After a new collection tube was added, 16ul of RNase-free water was added directly onto the membrane and the column was spun at maximum speed for 5 minutes. This step was then repeated. To quantify the cRNA the absorbance was read at 260nm and 280nm. The yield was determined using the following equation; Adjusted cRNA yield = RNAm – (total RNAi)(y) Where: RNAm = amount of cRNA measured after IVT (ug) Total RNAi = starting amount of total RNA (ug) y = fraction of cDNA reaction used in IVT
Hybridization protocol
The fragmentation buffer was made up as follows; 20ug cRNA, 6ul 5X fragmentation buffer, and the volume was made up to 40ul with RNase-free water. This solution was incubated for 35 minutes at 94oC, and then placed on ice. An aliquot was run on the Agilent Bioanalyzer to check the process had occurred Target hybridization The probe array (Affymetrix U133A gene chip) was equilibrated to room temperature immediately before it was used. Hybridization cocktail was made up of fragmented RNA (10ug), control oligonucleotide B2 (3nM) (3.3ul), 20X eukaryotic hybridization controls (bioB, bioC, bioD, cre) (10ul), Herring Sperm DNA (10mg/ml) (2ul), bovine serum albumin (BSA) (50mg/ml) (2ul) 2X hybridization buffer (100ul), dimethyl sulfoxide (DMSO) (20ul) and water to make the final volume 200ul. This was heated to 99oC for 5 minutes in a heat block. While the hybridization cocktail was heating, the 130ul of 1X Hybridization buffer was added to the array and it was incubated at 45oC for 10 minutes with rotation. The hybridization cocktail was transferred to a 45oC heat block for 5 minutes, and then was spun to remove any insoluble material. The buffer was removed from the array, and the hybridization cocktail (200ul) was added, and the array was placed in the Hybridization oven set to 45oC for 16 hours.
Scan protocol
The fluidics station was primed with Non-stringent wash buffer and Stringent wash buffer before use. After hybridization, the cocktail was removed from the probe array, and the array was filled with non-stringent wash buffer. Three staining solutions were made up and place onto the fluidics station. Staining solutions 1 and 3 were 600ul Streptavidin Phycoerythrin (SAPE) solution made up of 600ul 2X stain buffer, 48ul of 50mg/ml BSA, 12ul 1mg/ml SAPE, 540ul water. The 2nd stain solution is Antibody solution made up of 2X stain buffer (300ul), 50mg/ml BSA (24ul), 10mg/ml goat Immunoglobulin G (IgG) stock (6ul), 0.5mg/ml biotinylated antibody (3.6ul) and water (266.4ul). The fluidic protocol was as follows: Post hybridization wash #1 10 cycles of 2 mixes/cycle with wash buffer A at 30oC Post hybridization wash #2 6 cycles of 15 mixes/cycle with wash buffer B at 50oC
Description
CD38- sub-clones derived from a CLL patient with bimodal expression of the CD38 antigen
Data processing
Analysis of the microarray data was carried out on GeneSpring software (Agilent Technology). Firstly, any genes that were not present in at least one of the samples were removed from the gene list. Then any genes that had less than a 2 fold change were also removed. Statistical analysis was carried out (ANOVA test and Student’s t test) and a gene list produced after comparing the gene lists from the two tests and finding the genes present in both. The gene list was exported into Pathway Assist (Stratagene), where pathways were produced with any genes that were directly connected. Pathway assist connects to Pubmed (http://www.ncbi.nlm.nih.gov) and searches for any associations between the genes input. It then uses this information to build the pathway.
