Related Experiment Video
Updated: Jun 25, 2026
07:30
Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples
Published on: June 8, 2020
Analysis of variance components reveals the contribution of sample processing to transcript variation.
Douwe van der Veen1, José Miguel Oliveira, Willy A M van den Berg
1Laboratory of Microbiology, Fungal Genomics Group, Wageningen University and Research Centre, Dreijenplein 10, Building 316, 6703 HB Wageningen, The Netherlands.
Applied and Environmental Microbiology
|February 24, 2009
Summary
Designing DNA microarray experiments for Aspergillus niger requires understanding biological and technical variation. A quantitative real-time PCR (qPCR) approach identified day-to-day handling as the primary source of variation, informing experimental design.
Area of Science:
- Microbiology
- Molecular Biology
- Genomics
Background:
- Designing DNA microarray experiments necessitates understanding biological and technical variability in the model organism.
- Filamentous fungi like Aspergillus niger present unique challenges in experimental design due to inherent biological and technical variations.
Purpose of the Study:
- To establish a quantitative real-time PCR (qPCR)-based hierarchical experimental design for Aspergillus niger to determine sources of variation.
- To analyze the global transcriptional response to d-xylose using Affymetrix microarrays and identify differentially expressed genes.
- To provide parameters for optimizing the experimental design and data interpretation of microarray studies in filamentous cultures.
Main Methods:
- A quantitative real-time PCR (qPCR)-based hierarchical experimental design was employed.
- Analysis of variance components was used to quantify contributions of different processing steps to total variation.
- Affymetrix microarrays were utilized to analyze the global transcriptional response to d-xylose.
Main Results:
- Day-to-day handling and processing accounted for 68% of the total variation in the experimental setup.
- Fermentor vessel, cDNA synthesis, and qPCR measurement each contributed equally to the remaining variation.
- Twenty-four differentially expressed genes were identified in response to d-xylose, including enzymes for polysaccharide degradation and metabolism.
Conclusions:
- A defined experimental setup using qPCR and variance component analysis is a fast and reliable method to determine biological and technical variation.
- The study confirms that d-xylose signals the availability of complex polysaccharides to Aspergillus niger.
- Optimized experimental design parameters derived from this approach enhance the evaluation and interpretation of microarray data for filamentous cultures.
Related Concept Videos
02:01pre-mRNA Processing
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
02:01Pre-mRNA Processing
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
01:11Variability: Analysis
Measures of variability are statistical metrics that reveal the dispersion pattern within a dataset. They are pivotal in biostatistics, providing insights into the heterogeneity within health and biological data. Variability signifies the degree to which data points diverge from one another, helping researchers understand the potential range of values and associated uncertainty within the data.
The range is a simple measure of variability, indicating the difference between the highest and...
The range is a simple measure of variability, indicating the difference between the highest and...
01:32Pre-mRNA Processing: RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
01:21Sample Preparation for Analysis: Overview
Sample preparation is an essential step in the analytical process. It involves preparing a sample so that it can be analyzed accurately. The goal is to extract the analyte, the substance you want to measure, from the sample while removing any components that may interfere with the analysis. Sample preparation techniques vary depending on the physical state of the sample.
Bulk or large solid samples are typically reduced in size using grinding, crushing, or milling techniques to increase the...
Bulk or large solid samples are typically reduced in size using grinding, crushing, or milling techniques to increase the...
01:35Pre-mRNA Processing: Modification of pre-mRNA Ends
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...