18 Questions
What is the method of choice for high-throughput RNA expression analysis?
Microarray hybridization
What technique allows the detection of unlabeled cDNA sequences?
Gold nanoparticles technique
Which technology is reported to increase sensitivity by using gold nanoparticles rather than fluorescent tags?
MPSS analysis
What type of elements are arranged as spots in a grid pattern on a DNA microarray?
DNA elements
In DNA microarray technology, the arrayed targets are hybridized with what type of probe?
Complex probes with different sequences
What technique has been applied in whole-transcriptome analysis in Arabidopsis?
MPSS analysis
What is the role of the Bbv1 enzyme in the process described?
Chewing a small fragment from the cDNA
What determines the resulting four-base overhang in the process?
The cDNA sequence
What is the purpose of the adaptors in the process?
Annealing to cleaved cDNA
How does the encoding adaptor contribute to the process?
Allowing repetition of the process
What was used to determine the accuracy of the method described?
MPSS analysis on yeast cells
How many MPSS signatures were obtained from induced THP-1 cells during expression analysis?
Over 1.5 million
What is one key difference between eukaryotic mRNA and bacterial mRNA regarding polyadenylated tails?
Eukaryotic mRNA has a polyadenylated tail used for cDNA synthesis, while bacterial mRNA generally lacks this tail.
Why is selective priming with polyadenylated tails used for cDNA synthesis in eukaryotes?
To generate a labeled probe devoid of rRNA and tRNA.
Why did Richmond and colleagues use glass microarrays with a fluorescent probe in their study on E.coli transcriptional profiling?
To obtain more reliable and consistent results compared to nylon arrays with a radioactive probe.
How was the effect of isoniazid on Mycobacterium tuberculosis identified by Wilson et al. in 1999?
By exposing the bacteria to isoniazid and analyzing induced genes with a DNA array.
In bacterial gene expression studies, what has microarray hybridization been primarily used for?
To identify potential new drug targets.
What was one significant finding when E.coli was analyzed after exposure to heat shock by Richmond and colleagues?
The presence of rRNA did not interfere with the sensitivity of hybridization.
Study Notes
MPSS Analysis
- MPSS technique allows detection of unlabeled cDNA sequences, increasing sensitivity with gold nanoparticles instead of fluorescent tags.
- MPSS has been applied in several studies, including whole-transcriptome analysis in Arabidopsis and comparison of human cell lines.
DNA Microarray Technology
- DNA microarray technology allows parallel analysis of thousands of genes on a miniature device.
- Microarray hybridization is the method of choice for high-throughput RNA expression analysis.
- DNA microarrays comprise a series of DNA elements arranged as spots (features) in a grid pattern on a miniature solid support.
MPSS Process
- The initiating primer recognizes the DpnII overhang, and carries the recognition site for a type IIs enzyme, Bbv1.
- The cleaved cDNA is annealed to a set of 16 encoded adaptors, each recognizing a specific 4-nt overhang.
- Scanning of the bead after each round of hybridization reveals the 4-bp overhang, generating a sequence signature for each cDNA.
Accuracy and Applications
- The accuracy of MPSS was determined by analyzing early and late log phase yeast cells, with over 90% of signatures matching sequences in public databases.
- MPSS was used for expression analysis on human THP-1 cells, obtaining over 1.5 million MPSS signatures.
Eukaryotes and Bacteria
- In eukaryotes, mRNA has a polyadenylated tail, allowing selective priming of first-strand cDNA synthesis and generating a labeled probe devoid of rRNA and tRNA.
- Bacterial mRNA generally lacks a polyadenylate tail, so hybridization is carried out with total RNA.
Bacterial Expression Profiling
- Genome-wide transcriptional profiling has been used to study E. coli response to heat shock and lactose analog IPTG.
- Expression profiling has been used to identify potential new drug targets in bacteria, such as identifying genes involved in biochemical pathways.
Learn about the differences in mRNA structure between eukaryotes and bacteria, and how this impacts cDNA synthesis and hybridization. Explore the use of polyadenylated tails in eukaryotic mRNA and the genome-wide transcriptional profile of E. coli.
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