- Explain the function of a nucleic acid probe and describe the features that are required for its function.
- Outline the major steps in Northern blotting, RT-PCR, qPCR, microarray analysis and in situ hybridization. Explain the purpose of these techniques.
- Apply the above mentioned techniques to experimental questions (and know why to use each one)
Measuring Gene Expression
We often ask and measure expression of genes at the RNA level, and questions such as: “How much is expressed?”, “When is it expressed?”, or “Where is it expressed?” are often asked. There are many different techniques, and they have their own purposes. These techniques are Northern Blotting, Reverse Transcription-PCR (RT-PCR), Quantitative PCR (qPCR), Microarray Analysis, and in situ hybdridization.
RNA is easier to look at than a protein, and says what the functional product is that is being produced (not how much of it, but just that it is being produced).
Just because a gene is present in DNA doesn’t mean it is being transcribed and/or translated into a functional product. Testing RNA will determine what genes are actually being produced via transcription.
A lot of the RNA techniques will rely on probes because they allow for detection and visualization of the sequence of interest.
Probes a are short complementary nucleic acids, which are labeled to allow the detection of a sequence of interest in a complex mixture.
Probes can work in solution and on a solid substrate.
Probes are often labeled either radioactively, with a fluorescent tag, or a small molecules (e.g, digoxygenin, biotin)Credit: https://www.youtube.com/channel/UCxUHVv2k31uTOiCm4njuRfQ
Allows for the detection of specific RNA sequences (and their levels)
The first step is that the RNA is isolated from a sample and extracted. Then the RNA is processed via gel electrophoresis. The RNA is separated based on size, similar to the separation of DNA, then the RNA is transferred to a membrane where it becomes static.
The transfer consists of taking the membrane and placing it onto of the gel containing the RNA, then adding paper towel to the top of the membrane. Addition of the paper towel allows for capillary action to occur bring the RNA towards and onto the membrane.
The the RNA is hybridized with a probe for visualization.
Northern Blotting allows for questions to be answered such as:
- Is gene of interest expressed in sample?
- Is RNA of interest in sample?
- is RNA of interest present in bands?
It detects a specific transcript and its amount, size, and splicing products. This can be done in different developmental time points, and tissue types.
It is the tangent of Southern Blotting named after Sir Edwin Southern.Credit: https://www.youtube.com/channel/UCFAQwnv2kGxl_2BOrI6790A
Remembering Mnemonics: SNOW DROP:
O cancels O
Reverse Transcription PCR (RT-PCR)
Reverse transcription is the reverse process of transcription which means that RNA is a template to produce DNA.
Reverse transcription is done by the addition of a reverse transcriptase enzyme, and a polyT primer. The polyT primer complementary base pairs to the polyA sequence. Since only eukaryotic cells contain polyA tails in their mRNA this specific application is only possible in eukaryotic cells.
The annealing of the polyT primer to the polyA tail allows the reverse transcriptase enzyme to anneal and produce a complementary DNA sequence to the mRNA.
Once the complementary DNA sequence is produced. PCR application is done, and will answer the question “Is the seqeunce of interest present?”
If not= No product will be present
PCR is performed normally, with the exception that DNA is single stranded post reverse transcription is processed. This is done with gene specific primers, and Taq (Thermos aquaticus) DNA polymerase.Credit: https://www.youtube.com/channel/UCQ9Z0a4SZdsn-WUyyloGn6A
What does RT-PCR tell us?
Whether the RNA of interest is present, and that’s all folks!
Most experiments are only amplifying small fragments of the RNA, and not the whole gene. Not because it can’t, just because the way the experiment is styled to do. PCR is faster and easier to do than Northern Blotting. Also more quantitative than Northern blotting, but does convey less information about the transcript size because it typically does not amplify the whole transcript.
Quantitative PCR (qPCR)
Quantitative PCR, unlike the standard equivalent (which only detects products after the completion of all cycles), detects products ar each cycle.Credit: https://www.youtube.com/channel/UCL2CiNwEBjt-JOFB8-uIRyw
Standard vs Quantitative PCR
A standard PCR is done by running the product on to the gel, and determine whether amplification has happened
Quantitative PCR after every cycle, we determine how much product is present and then we take a picture.
PCR is non linear by the end of the reaction, whereas qPCR can measure products during the linear phase.
- It is plateau since primers/ nucleotides run out
Data is compared in exponential phase to a standard curve, and this allows us to calculate the template concentration base on the shape of the curve.
It allows us to know the starting / differing amount of products after each cycle. Different in amplification by taking the DNA out early on.
If the DNA is unknown, you should get data points from ech cycle, however should still finish each cycle.
The PCR reaction is monitored via fluorescence, which quantitates the amount of DNA at the beginning, but not the whole concentration for the sequence of interest.
After each PCR cycle the fluorescence is detected, and used to calculate the template DNA concentration.
The mechanism behind this is by using SYBR, which intercalates into the double-stranded DNA (dsDNA). It is important to know that fluorescence will only occur when it is banded to a double stranded DNA, thus, after each cycle SYBR will bind to the product of each PCR cycle.
Coupling reverse transcription and qPCR will result in the sensitive quantitation of RNA levels. This means that the we are able to quantify the amount of mRNA that is present, by RT-PCR reverse transcribing the mRNA to cDNA (complementary DNA), and then qPCR to quantify the concentration of cDNA present in the sample.
RT-PCR is performed, but on a scale of the whole whopping genome!
Gene specific DNA sequences are printed onto a glass slide, and there are DNA imprints on each spot. They have thousands of copies for a specific gene.
mRNA is reverse transcribed, and then labeled all the mRNA. The products as a result are hybridized to the “chip” of the microarray (as the chip is complementary to the cDNA, and identical (U’s exchanged for T’s) to the mRNA).
Once this is done the mRNA is then measured by means of fluorescence, and will indicate the amount of the specific gene that is present in the sample.
They are usually comparative, and the ratio between the two samples, for example, a tumour cell and a normal cell both have the sequence for gene A so it comes back as yellow (red + green), however normal cells express gene B more (which will show up green) and tumour cells express gene C more (which will show up red).
Examples: Tumor vs normal tissue, drug vs no drug treatment, embryo vs adult, muscle vs brain.Credit: https://www.youtube.com/channel/UCxUHVv2k31uTOiCm4njuRfQ
in situ (in the organism/tissue) hybridization
Tells us where the transcript is expressed.
A labelled probe is hybridized to the organism/tissue. This tells us where the probe is present (where RNA is present due to complementary base pairing), and allows for visualization.Credit: https://www.youtube.com/channel/UCkCxe9tf9J9R0XNz_Tcp_-A