RNA QC Solutions on the 2100 Bioanalyzer
RNA QC Solutions on the 2100 Bioanalyzer
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Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.
While microarray technology has offered new insights into gene expression and biological processes, the efficacy of analysis is directly related to the quality and integrity of the starting RNA, particularly when using direct labeling procedures through reverse transcription. Agilent’s 2100 Bioanalyzer offers a microfluidic-based tool for the analysis of both nucleic acids and proteins, to examine the ability of a sample to provide high quality data for microarray analysis. Sherry Grissom and colleagues (2005) examined the capabilities of the 2100 Bioanalyzer for assessing the quality of directly labeled fluorescent cDNA prior to microarray hybridization using varying amounts of RNase to simulate RNA degradation in a novel assay. The group shows the strength of this assay when used in conjunction with the 2100 Bioanalyzer in determining the relative amounts of cDNA obtained from a direct labeling reaction. Results indicate that utilization of this method helps to prevent costly mistakes associated with the hybridization of poor quality direct labeled products to expensive arrays.
Figure 1. RNA and Cy5-dUTP labeled cDNA measured by the Agilent 2100 Bioanalyzer.
Electropherogram images (one representative experiment) of RNAs treated with RNase as follows: No RNase (A, pink), 0.01 ng ml-1 (B, blue), 0.1 ng ml-1 (C, red), 1 ng ml-1 (D, green). Overlayed electropherogram image of Cy5-dUTP labeled cDNA (E, colors as described above, one representative experiment). The first peak (as shown by an arrow) from the left in all electropherograms is a 50 base pair marker present in the sample buffer (M). The second peak (as shown by an arrow) that is present only in Cy5 samples is free Cy5-dUTP (Cy5) and measures approximately 150 nucleotides (data not shown). Though it is a single nucleotide, the Cy5 modification is large, which could explain its delayed migration and detection.
Figure 2. Comparison of Bioanalyzer assay with conventional gel analysis.
(A) cDNAs were separated based on size using a 1% agarose gel. Red fluorescence (650 long pass emission filter) was measured using a Storm phosphorimager and quantitated using IMAGEQuant. (B) "Gel-like" images obtained from the Bioanalyzer are digital images rendered from conversion of electropherogram fluorescence traces using the Bioanalyzer software. Both images are the same sample from one representative experiment ran in parallel.
Figure 3. Practical application of the Cy5-cDNA Bioanalyzer assay.
(A) Overlayed electropherogram image of Cy5-dUTP labeled cDNA obtained from control RNAs (MCF7 and HACAT, red and blue traces, respectively) and from test samples (Test #1 and Test #2, brown and purple traces respectively). First and second sharp peaks are as described in legend for Figure 1. (B) Average microarray intensity for MCF7, Test 1 and Test 2. Error bar is standard error from three arrays. Test samples were hybridized to only one microarray each.
Original Research Paper:
Title: A qualitative assessment of direct-labeled cDNA products prior to microarray analysis.
Authors: Grissom SF, Lobenhofer EK, Tucker CJ.
Journal: BMC Genomics. 2005 Mar 11;6(1):36.
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The RNA Integrity Database
Use the new RNA Integrity Database (RINdb) as an additional tool for RNA QC to validate your protocols and sample quality by comparing your results to those of your peers for hundreds of tissue samples.
For research use only and not for use in diagnostic procedures.