RNA Analysis Flashcards
What is RT-PCR?
PCR amplification of cDNA samples
How is RT-PCR performed?
The entire cellular mRNA is reversely transcribed to cDNA, and then gene or mRNA specific RT-PCR is carried out
How can RNA be analyzed?
Most DNA analysis techniques can be adapted for RNA analysis, either directly or through reversely transcribing complementary DNA (cDNA)
How does first strand cDNA synthesis work using the Oligo(dT) primers?
1) The Oligo(dT) primer anneals to the polyA tail of the mRNA
2) Synthesis
But, synthesis may not go all the way to the end of the mRNA strand, and there can be an over-representation of the 3’ end
Many will miss the 5’ cap, which means the start of the transcript is missed.
How does first strand cDNA synthesis work using random hexamer primers?
The primers anneal at random sites on the mRNA, and there will be good coverage of entire transcript, but certain pieces will be missed
Can transcribe the 5’ end regions that are unreachable by the oligodT primer, or for cDNA synthesis of species that do not have a polyA tail
Hexamer primers do not require the polyA tail
How does 5’ RACE work?
Capture the 5; end of mRNA and make the full length cDNA clone
mRNA –> cDNA
1) Reverse transcriptase extends incomplete cDNA (start at 3’ end of mRNA) by designing a short primer is to anneal to the 3’ end of the incomplete cDNA
2) Terminal Transferase, RNase H which removes the mRNA strand
3) Add a string of nucleotides to the 3’ end of cDNA, often C nucleotides
4) Add DNA polymerase and an oligodG primer which anneals to the C nucleotides at the 3’ end and a 5’ to 3’ second strand based on cDNA is made
5) PCR
Why does the 5’ end need to captured in cDNA?
5’ end could have exons and be part of the ORF
Without capturing the 5’ end, pieces of the sequence could be missed
mRNA is just comprised of exons, no introns, no promoters, UTR is present
How are cDNA libraries made?
1) RT of cellular mRNA
2) Convert first strand cDNA to blunt ended double stranded cDNA
3) Use a linker/adaptor to batch clone these fragments into expression plasmids
What is the RNase protection assay?
Some RNAses only degrade single stranded RNA
This can be used to map the 5’ and 3’ ends of the transcripts
Quantify relative abundance of specific transcripts
Map intron/exon boundaries and identify splicing variants
How does the RNAse protection assay work?
1) Design a radiolabeled anti-sense RNA (which is based off the genomic region) that has some part of the promoter region
* mRNA is present for the assay** –> use mRNA for hybridization
2) Hybridization of anti-sense RNA probe with mRNA
3) Degrade ssRNA with RNAse: Some parts of the radiolabeled RNA will be degraded because anti-sense to introns and mRNA does not have introns, so will not base pair, also the promoter region of genomic DNA will also not base pair with mRNA, because promoter is not included in mRNA
4) Detect remaining probe by electrophoresis
How is the RNAse protection assay used to map splicing events?
Design anti-sense RNA probe (based off of the DNA genomic region)
No splicing: Entire probe is base paired, entire probe is protected, and because entire probe is protected, nothing will be degraded. (1 fragment)
Splicing: The intron is not base paired, and therefore is looped out, and degraded by RNAse. As such, there are two fragments, exon 1 and exon 2, as the intron in the middle was degraded
Run a gel to see fragments
What is an example of a RNAse protection assay?
Adenovirus E1A RNA splicing
Digest with RNAse that only cleaves single stranded RNAs, double stranded RNAs are protected from digestion
