Intro to SRP Flashcards
How do you look at mRNA accumulation within cells/tissues/embryo?
prepare probes that is complementary to mRNA
How do you make this probe?
Make cDNA from mRNA using reverse transcription. The single stranded DNA that results from reverse transcription is then going to be made double stranded by PCR, which also Amplifies the cDNA so there is enough copies of it to use in lab. Once the double stranded cDNA is generated, it can be used to make anti-sense RNA, which serves as the probe.
upon making the anti-sense RNA, DIG molecules will be incorporated such that we can detect them with antibodies.
What is the sense and anti-sense strand?
mRNA in 5’ to 3’ direction represents sense strand of RNA molecule.
we can generate anti-sense cDNA using the mRNA template using reverse transcriptase to reverse transcribe that mRNA (going against central dogma).
so using mRNA as its template, will synthesise a single stranded cDNA (process in 5’ to 3’ direction).
What is genomic DNA expressed mainly regulated by?
promoters - typically upstream of a gene but can be downstream of it or anywhere else along the DNA
Explain genomic DNA transcription
- Involves the unwinding of the DNA double helix, the attachment of RNA polymerase to the anti-sense, and using this strand as a template from a transcript of RNA in the 5’ to 3’ direction (RNA polymerase reads in the 3’ to 5’ direction). The RNA generated is therefore in the sense orientation.
- The RNA is then processed with this including the removal of introns as well as capping at the 5’ end and the addition of a polyA tail at the 3’ end/ This mature mRNA is then exported to the cytoplasm and then used as a template for translation as part of a ribosome complex.
What are the two ways gene expression can be looked at?
In situ hybridisation that looks at the localisation of mRNA within tissue or via immunostaining through the use of antibodies.
Explain the steps involved in in situ hybridisation
- Involves the recognition of particular mRNA via a probe with this probe also being attached to DIG (digoxigenin) that has been added to the probe in the lab.
- The probe is then hybridised to the particular mRNA of interest and anything else that has not bound to any mRNA is then washed away
- The sample being analysed is then incubated with an antibody that recognises the DIG molecule. This antibody has AP attached to it.
- Any antibody that has not attached is then washed away.
- Following this, a substrate that turns dark purple upon exposure to AP is added to the sample
- The result is regions that contain the mRNA of interest are highlighted by the dark purple staining.
How can immunostaining be used to look at gene expression?
An antibody that recognises an antigen from the protein coded by the gene of interest is added to the sample with this antibody then allowed to bind to its antigen.
To amplify the signal, a second antibody that recognises the immunoglobulin part of the first antibody is added and this second antibody will have a flourescent molecule attached to it (flourescein if green and rhodamine if red)
The different coloured flourescent molecules allows for the analysis of the expression 2 or more proteins at the same time.
This process of forming cDNA is done by using a strand of…
thymidine molecules to attach to the polyA tail of the mRNA with reverse transcriptase then completing the anti-sense transcript using the mRNA strand as the template.
The cDNA is initially single stranded so is first made…
…double-stranded before it is amplified by PCR
This process is actually the part where the specific transcript of the gene of interest is differentiated from the other genes. This is due to the strand of Ts does not bind specifically to any particular gene (polyA tail is the same for all genes).
The primers used in PCR however are specific meaning this is the point where..
the transcript of the gene of interest is differentially amplified over the other genes
The primers also contain an RNA polymerase binding site known as..
T3
This double-stranded DNA (cDNA) is then made into single strands with the sense strand used to generate anti-sense RNA and this is only possible due to T3 allowing RNA polymerase to bind to the sense strand.
The anti-sense RNA generated is then attached to DIG to form the probe to be used. The way this occurs is one of the nucleotides is replaced by a modified nucleotide that has DIG attached to it. The process of incubation within the sample of interest (e.g. body of zebrafish) followed by hybridisation then follows as described above.
