We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 3

1.
Figure 3

Figure 3. From: New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community.

Periodic gene expression. Relative mRNA abundance for 47 periodically expressed reference genes at eight time-points covering the intraerythrocytic development cycle of P. falciparum strain 3D7. Pearson’s coefficient of correlation (r) is indicated when compared to corresponding time-points of a prior published 3D7 time-course [11].

Björn F C Kafsack, et al. Malar J. 2012;11:187-187.
2.
Figure 2

Figure 2. From: New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community.

High consistency of log-ratio measurements across a 20-fold range of hybridized material. Heat map of mean-centered log2(Cy5/Cy3) ratios for six genes representing a 32-fold intensity range (see Figure 1A) across a 20-fold range of hybridized material. For each gene the maximum percent fold-difference (Δmax) between any two of the triplicate averages is also shown. As a final assessment of array performance relative transcript abundances at eight IDC time points were measured at 6-hour intervals. At each time-point P. falciparum strain 3D7 total RNA was isolated, reverse transcribed, and Cy5-coupled. These eight samples were each hybridized to one of the eight arrays on a single 8x15K slide, along with an equal amount of Cy3-labelled reference pool. To illustrate correspondence of these results with previously published work, 47 periodically expressed reference genes were chosen that peak in expression within successive three-hour windows [9]. This reference set (available as a table in Additional file 7) can be used for easy visualization of a variety of time-course attributes such as synchrony, progression through the IDC, correct ordering of time-points, etc. As expected, the abundance profiles of these reference transcripts reproduced the characteristic “barber-pole” pattern of the IDC transcriptional cascade (Figure 3) and mirrored existing results at corresponding time points (median Pearson’s = 0.95). [11,49]. The results remained consistent when extending this comparison genome-wide (median Pearson’s r = 0.83).

Björn F C Kafsack, et al. Malar J. 2012;11:187-187.
3.
Figure 1

Figure 1. From: New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community.

High overall consistency of log-ratio measurements across a wide range of transcript abundance. Normalized Cy3 (white), Cy5 (black), and log2(Cy5/Cy3) (grey) intensities from triplicate self-hybridization experiments for (A) Six genes with average intensities representing a 32-fold range (I = MAL7P1.89, II = PF14_0419, III = PF11_0528, IV = PF07_0118, V = MAL8P1.139, VI = PF11_0506) and (B) the 18 probes targeting MAL8P1.139 along with the probe average. (Error bars represent SEM). Non-feature background levels for the 8x15K arrays are very low with 98.6 % of probes yielding both Cy5 and Cy3 signals at least 2.5 standard deviations above background (data not shown). This needs to be taken into account during analysis, as weak non-specific binding of features targeting genes known to not be expressed in some samples occasionally produce very low signal that is nevertheless above non-feature background. In order to assess the minimum amounts of total RNA starting material and dye-labelled cDNA required, cDNA was generated from a pool of total RNA harvested at various stages of the IDC. As little as 500 ng of total RNA starting material yielded 460 ± 13 ng of amino-allyl labelled cDNA and 241 ± 4 ng of dye-coupled cDNA (Additional file 5) with 24 ± 0.4 fmol/ng of dye-incorporation. To test the minimum amount of cDNA required for hybridization, two separate pools of cDNAs with Cy3 and Cy5 were labelled respectively and hybridized a diminishing series of equal amounts from each pool (1,000 ng, 500 ng, 250 ng, 100 ng, 50 ng). Again examining the same set of genes representing a wide abundance range (see Figure 1A), only small differences in the Cy3/Cy5 log ratio measurements was observed across this range of hybridized material with the maximum fold-difference between any two of the 15 measurements for a given gene being 1.35-fold (Figure 2). No significant change in the number of transcripts with signal intensities called as “well above background” by Agilent Feature Extractor Software was observed when hybridizing decreasing amounts material (Additional file 6A). Furthermore, both Cy5 and Cy3 gene signal intensities array-wide correlated very highly (r > 0.96, Additional file 6B) across the dilution series down to 100 ng, and even at 50 ng of hybridized material gene signal intensities matched the 1000ng sample closely (r > 0.82, Additional file 6B). Thus, the amount of dye-coupled cDNA obtained from as little as 500 ng of total RNA is sufficient for performing two to four hybridization experiments.

Björn F C Kafsack, et al. Malar J. 2012;11:187-187.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk