PMC:15026 / 36809-39331 JSONTXT

{"project":"2_test","denotations":[{"id":"11178248-8828038-44513666","span":{"begin":273,"end":275},"obj":"8828038"}],"text":"Preparation of complex probes, multiplex PCR and differential screening of candidate genes\nIn this technique, nylon filters dotted with candidate genes are hybridized with complex cDNA probes from different cell populations, to compare expression levels (see, for example [76]). Originally developed and validated by SANOFI-Recherche (unpublished data), the nylon filters contain 91 rat candidate genes (PCR products), including signaling molecules and transcription factors (Figure 4). These cDNAs were selected with the idea of defining a panel of genes whose expression is likely to be modulated in response to a proliferative signal. To increase the sensitivity of the approach, the mRNAs of interest are co-amplified by reverse transcription polymerase chain reaction (RT-PCR), using primers specific for the 91 candidate genes (the multiplex PCR step), before their use as hybridization probes (Figure 3a). Under these conditions, moderate PCR amplification allows the detection of weakly expressed genes and the evaluation of their differential expression in a semi-quantitative manner. mRNAs are amplified by RT-PCR, and the number of PCR cycles is adapted to their relative abundance in the population tested (16, 21, 24 or 26 cycles are performed for the detection of relatively abundant, moderately expressed, weakly expressed and very weakly expressed transcripts, respectively). For each cell population analyzed, the multiplex PCR products are then mixed, radiolabeled by random priming with α-32P-labeled dCTP, and used as hybridization probes against the 91 candidate genes. Hybridization signals are quantified by densitometry (Visiomic) for each of the different populations. During the development of this technique, control studies involving repeated hybridizations with replicate filters showed minimum variation of signal response (data not shown).\nThe efficiency of the technique was controlled by including rabbit α and β globin cDNAs on the nylon membranes along with the candidate genes. Defined amounts (50 or 150 ng) of the globin cDNAs were also added to the cDNAs mixtures before the multiplex PCR step. As expected, when these different amounts of rabbit globin cDNAs were added to the complex cDNAs, spots of different intensities were obtained (Figure 3b), indicating that this approach could detect at least a threefold difference in mRNA expression between two cell populations.\nFigure 4 shows the list of the candidate genes analyzed and indicates their expression pattern in Nb2 cells."}

{"project":"Colil","denotations":[{"id":"T85","span":{"begin":273,"end":275},"obj":"8828038"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Preparation of complex probes, multiplex PCR and differential screening of candidate genes\nIn this technique, nylon filters dotted with candidate genes are hybridized with complex cDNA probes from different cell populations, to compare expression levels (see, for example [76]). Originally developed and validated by SANOFI-Recherche (unpublished data), the nylon filters contain 91 rat candidate genes (PCR products), including signaling molecules and transcription factors (Figure 4). These cDNAs were selected with the idea of defining a panel of genes whose expression is likely to be modulated in response to a proliferative signal. To increase the sensitivity of the approach, the mRNAs of interest are co-amplified by reverse transcription polymerase chain reaction (RT-PCR), using primers specific for the 91 candidate genes (the multiplex PCR step), before their use as hybridization probes (Figure 3a). Under these conditions, moderate PCR amplification allows the detection of weakly expressed genes and the evaluation of their differential expression in a semi-quantitative manner. mRNAs are amplified by RT-PCR, and the number of PCR cycles is adapted to their relative abundance in the population tested (16, 21, 24 or 26 cycles are performed for the detection of relatively abundant, moderately expressed, weakly expressed and very weakly expressed transcripts, respectively). For each cell population analyzed, the multiplex PCR products are then mixed, radiolabeled by random priming with α-32P-labeled dCTP, and used as hybridization probes against the 91 candidate genes. Hybridization signals are quantified by densitometry (Visiomic) for each of the different populations. During the development of this technique, control studies involving repeated hybridizations with replicate filters showed minimum variation of signal response (data not shown).\nThe efficiency of the technique was controlled by including rabbit α and β globin cDNAs on the nylon membranes along with the candidate genes. Defined amounts (50 or 150 ng) of the globin cDNAs were also added to the cDNAs mixtures before the multiplex PCR step. As expected, when these different amounts of rabbit globin cDNAs were added to the complex cDNAs, spots of different intensities were obtained (Figure 3b), indicating that this approach could detect at least a threefold difference in mRNA expression between two cell populations.\nFigure 4 shows the list of the candidate genes analyzed and indicates their expression pattern in Nb2 cells."}