A reference pool was created from total RNA isolated from nine different lymphoma cell lines (RNA control pool prepared at NIH) using TRIzol extraction.
Label
Cy3
Label protocol
Total RNA from the reference pool was labeled with Cy3 dUTP in a first strand cDNA reaction.
tissue: bone marrow phenotype: CD19-CD10-CD34+CD38-
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from 10,000 - 80,000 cells of each of the sorted cellular population (naive BC, centroblasts, centrocytes, memory B) with Absolutely RNA® Microprep Kit (Stratagene) followed by 2 rounds of linear amplification: in a 10 ul reverse transcription reaction, total RNA was transcribed by priming with a dT/T7-primer. Second strand synthesis was initiated by RNase digestion. The purified double stranded cDNA served as the template for the first round of in vitro aRNA transcription (MEGAscript® T7 Kit, Ambion Inc.). For the subsequent round of amplification, the first strand cDNA was primed with random hexamers. The synthesis of the second strand was initiated by annealing a dT/T7-primer to the aRNA:cDNA heteroduplex with partially digested aRNA. After purification, a second round of in vitro transcription was performed. The quality of the aRNA was always detected by the Agilent 2100 bioanalyzer.
Label
Cy5
Label protocol
Antisense RNA (5 ug) from an experimental sample was labeled with Cy5 dUTP in a first strand cDNA reaction.
Hybridization protocol
Samples were hybridized to the arrays as described in Alizadeh A. et al, Nature 403(6769):503-11.
Scan protocol
Fluorescent array images were collected for both Cy3 and Cy5 with a Axon GenePix Scanner (Axon Instruments) and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.
Description
none
Data processing
After background correction and removal of flagged values, data was normalized based on a global median normalization factor, and log base 2 (Cy5/Cy3) expression ratios were calculated to produce the values given in the data table.
