pH-Stat Titration Allows the Continuous Determination of Ribonuclease A Activity toward Cytidine 2′,3′-Cyclic Monophosphate at High Substrate Concentrations
✍ Scribed by Jens Köditz; Renate Ulbrich-Hofmann
- Publisher
- Elsevier Science
- Year
- 2002
- Tongue
- English
- Weight
- 69 KB
- Volume
- 305
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
fluorescent labeled NTPs and labeled, short nucleic acids were recovered, and the unlabeled NTPs and nucleic acids were discarded. The reverse-phase column-purified labeled NTPs were further purified with size fractionation using membrane filters in order to separate the NTPs from the higher molecular weight, labeled nucleic acids. The purification protocol took approximately 4 h and provided an estimate yield of 60% according to the starting material. The purity of the recovered NTPs was confirmed by HPLC (data not shown). On HPLC chromatograms no impurities could be observed.
The recovered fluorescently labeled Cy5-dCTP and Cy3-dCTP were used for microarray analysis. Human heart amplified, antisense RNA (aRNA) was used as a template for synthesis of labeled cDNA during RT with standard protocols. As a control experiment the same template was labeled with commercially available Cy5-dCTP or Cy3-dCTP. cDNA labeled with recovered Cy5-dCTP was mixed with commercial Cy3-dCTP-labeled cDNA and hybridized onto a human microarray, and normalized, average signal distribution of 6400 spots (3200 data points) was determined. The same experiment was carried out with recovered Cy3-dCTP and commercial Cy5-dCTP-labeled cDNA. After data analysis all the lowconfidence data derived from spots with high local background caused by dust particles or other impurity of the slide or from those exhibiting very low intensities were eliminated. Figure 1 demonstrates the tight distribution of data points deriving from the normalized ratio of intensities from commercially available Cy3-dCTP labeled cDNA and intensities from recovered Cy5-dCTP labeled cDNA. The color flip experiment gave the same tight distribution (data not shown). This means that purified NTPs did not distort the expression results and confirms that recovery can be performed without any risk in microarray experiments. Using recovered dye-labeled NTPs the expenses of microarray experiments can be decreased, especially when large number of samples are intended to be labeled and analyzed.