ABSTRACT
Fluorescent Linear Labelling technique is an effective, single-tube and linear amplification method. The sample cDNA was synthesized from total RNA, and was labelled antisense cRNA from double-stranded cDNA with T7RNA polymerase; it can be used for hybridization to oligonucleotide biochip. Fluorescent Linear Labelling Method can result in 50- to 100-fold higher degrees of amplification, and has been shown to retain information on transcript abundance, thus making it an efficient, robust technique for fluorescent labelling on biochip.
Subject(s)
Humans , Fluorescent Dyes , Chemistry , Oligonucleotide Array Sequence Analysis , Methods , Staining and Labeling , MethodsABSTRACT
BACKGROUND: Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells. Without effective treat ment, individuals in the indolent, chronic phase (CP) of CML will undergo blast crisis (BC), the prognosis for which is poor. Therefore, it is important to clarify the mechanism underlying CML from a whole-genome perspec tive. OBJECTIVE: To investigate the gene expression profile of bone marrow mononuclear cells from CML with Applied Biosystems Expression Array System.DESIGN: Observation and controlled analysis.SETTING: Institute of Gene Engineering, Southern Medical University PARTICIPANTS: Samples of two cases of bone marrow (a chronic myeloid leukemia patient and a healthy person).METHODS: This experiment was conducted at the Institute of Gene Engineering, Southern Medical University from October 2004 to September 2005.The total RNAs were extracted and purified from bone marrow mononuclear cells derived from a CML patient and a healthy person. mRNAs were purified using an oligo (dT)-cellulose mRNA purification kits and labeled using reverse transcription, in vitro transcription (RT-IVT), then hybridized with microarray. Gene expression differentiation of the bone marrow mononuclear cells were examined by ABI 1700 Chemiluminescent Microarray Analyzer. Reproducibility of microarray results was assessed by comparing data sets obtained from the same sample and analyzed by two different arrays.MAIN OUTCOME MEASURES: ①Assessment of quality of total RNA and labled cRNA. ②Reproducibility of microarray. ③ Hybridization of array.④Results of semi-quantitative reverse transcription-polymerase chain reaction RESULTS: ①Using statistical data analysis tools, we identified 6 706 genes that were up- or down-regulated in CML patient compared with the healthy person. In these genes, we found that 17 genes were up-regulated while 51 genes were down-regulated among 68 genes closely related to CML. ②most differentially expressed genes in C/EBPalpha mediated path way and CD40L signaling pathway had reduced expression. ③Good repro ducibility of microarray was confirmed by analysis of correlation and detection concordance in technical replicates. The correlation coefficient of the detectable probe in technical replicates was 0.991 for the CML patient and 0.988 for the healthy person. ④The results of semi-quantitative RT-PCR experiments supported the reliability of our microarray analysis.CONCLUSION: By comparing expression patterns of CML with those of the healthy person, we identified a large number of genes that, were up- or down-regulated in CML patients. These data should provide useful information for finding candidate genes whose products might serve as molecular targets for treatment of CML patients.
ABSTRACT
Objective To report a new method of fluorescent labeling technique in microarray studies: universal primer U2 labeling( UPL). The efficiency was compared of the UPL with that of random primer, restriction display labeling method and the reverse transcription coupled random primer spiking labeling method(RT-PSL). Methods Influenza viral RNA was labeled with both UPL and the conventional random primer labeling method as well as two other more laborious labeling methods( RD-direct and RD-incorporate), and hybridized with influenza virus oligonucleotide microarrays. The signals extracted from the microarrays were analyzed using SPSS 10.0 software. Results The fluorescent intensity, signal-to-noise ration(SNR), true positive ratio(TPR) of probes and labeling reproducibility of UPL were demonstrated to be higher than those of the Random primer approaches.Conclusion These results established that UPL is a valid new labeling protocol, which may have wide applications in the research and development of the microarray technology.