Theme 3- Module 4 Flashcards
What is in situ hybridization used for?
To identify which embryonic cells/tissues express a gene that play an important role in the embryonic development of the organism
What does in situ hybridization do?
Finds WHERE the corresponding mRNA that is transcribed from that gene can be found during development in the intact organism
How does in situ hybridization work?
Fluorescently labelled short single-stranded segment of DNA or RNA binds/hybridizes in a complementary fashion to the target mRNA molecule
What can we do to attain a better understanding of temporal differences that may exist in gene expression during development?
Carry out and compare (spatial) situ hybridizations throughout the different stages of development
What is the advantage of using DNA microarray techniques instead of in situ hybridizations?
With DNA microarrays, you can examine the expression of thousands of genes at once
In situ hybridization is useful to assess the gene expression levels of a FEW genes of interest
How do DNA microarrays work?
Set up glass slides that have tiny spots containing a known DNA sequence or gene
DNA molecules act as probes to detect gene expression
What is the transcriptome?
The set of mRNA transcripts that are expressed by various genes
What are oligonucleotides?
Short fragments of nucleic acids
How can we find out which genes are involved in transformation of a normal cell to a tumour cell?
Grow both cell types in culture
Isolate the gene products or mRNA that is transcribed by these two cell types
Undertake a DNA microarray analysis.
Once the mRNA has been isolated from normal cells and tumour cells, what are they used for in the proceeding DNA microarray analysis?
They serve as templates for making complementary cDNA molecules to the mRNA
Which enzyme is responsible for making complementary cDNA molecules to the mRNA?
Reverse transcriptase enzyme
What do they use to make it easier to identify the different cDNA during the microarray analysis?
Different coloured fluorescent nucleotides that become part of the newly synthesized cDNA molecule
Describe the main steps of preparing cancerous and normal DNA for microarray analysis
Isolate mRNA from cancer cells and normal cells
Reverse transcription to cDNA with fluorescent nucleotides
Combine equal amounts of labelled cDNA from normal cells and from cancer cells
Apply mixture to microarray chip
Test for hybridization
How can you know a particular gene is active based on the microarray analysis?
Active genes produce many molecules of mRNA
We will therefore have more labelled cDNA molecules available after reverse transcription
(which will be able to hybridize to the DNA on the microarray chip and produce bright spots)
Why is it important to have an equal mixture of the labelled cDNA of both the normal epithelial cells and breast carcinoma cells?
They need to compete for the synthetic complementary DNA fragments in the spots of the microarray chip.
Normal cDNA were labelled with green and cancer cDNA were labelled with red.
If a fluorescent spot is green for example, that means:
a) the specific gene in that spot is upregulated in normal cells and downregulated in carcinoma cells.
b) the specific gene in that spot is downregulated in normal cells and upregulated in carcinoma cells.
a) the specific gene in that spot is upregulated in normal cells and downregulated in carcinoma cells.
Why are the spots brighter if the gene is more active?
If the gene is active, it produces more mRNA, which leads to brighter spots.
If there is no fluorescence at all in a particular spot, what does that indicate?
That the gene may be inactive
How can computational software provide further insight into groups of genes that may participate in common cellular processes at common times?
It can further analyze the data from multiple microarrays and cluster genes that show similar expression patterns
What is the purpose of short, noncoding regulatory double stranded RNA molecules?
To activate RNA interfering machinery which then interferes with mRNA translation
Give two examples of small regulatory RNA
microRNA
siRNA
How are both types of small regulatory RNA processed?
Transcribed from protein-encoding genes
Form hairpin loops due to complementary base-pairing
Loops are then cleaved (via enzymes) into smaller, single stranded mRNA fragments
Becomes incorporated as part of a (or RISC complex)
After activating the RISC complex, how do microRNAs inhibit translation?
Contain sequences that are complementary to specific target
Will bind in a NON-EXACT manner to the target mRNA sequence and the proteins within the RISC complex then inhibit translation
What does the RISC complex stand for?
RNA-induced silencing complex
