John M. S. Bartlett.pdf - Bio-Nica.info

RNA Arbitrarily Primed PCR 251
Fig. 2. Scatter plot of fluorescent intensity of RAP-PCR products analyzed by microarray
hybridization. Samples were RAP-PCR products (1 µg) from duplicate fingerprint reactions labeled
using Cy3-dCTP (green fluorescence) or Cy5-dCTP (red fluorescence), random primer and
Klenow as described. The microarray contained 6400 double spotted cDNAs of known identity.
400 mL of each solution and place them in three different glass chambers. To minimize
the transfer of solutions, a different container should be used each time and only the slides
should be transferred. The solution should then be spun down 5 min to dry the slides.
3.7. Analysis of Microarray Data
Analysis of the microarray hybridization was performed using a ScanArray 5000
or equivalent instrument to monitor fluorescent signal. Data was gathered first from
the Cy5 dye followed by scanning of the Cy3 fluorescent signal. Typical instrument
settings were laser power at 50 and photomultiplier tube at 100. Microarrays were
scanned using 10-µm resolution. Quantification of microarray data was performed
using QuantArray software or an equivalent software package. The results are shown
in a scatter plot in Fig. 2. More than 95% of the signals that are significant relative to
negative controls from salmonella are within twofold.
4. Notes
1. Knowing the total RNA concentration is critical for RAP-PCR. cDNA is synthesized
at several concentrations ranging between 1000 ng and 125 ng of total RNA per 10-µL
reaction. At lower concentrations of input total RNA there sometimes arise spurious PCR
products, which overwhelm the amplification step. Also, DNA contaminants are monitored
by the inclusion of a reverse transcriptase-free control in initial RAP-PCR experiments.
This reaction is set up exactly as a RAP-PCR, but reverse transcriptase is omitted and
replaced with water. The result should be a gel lane with no bands.