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mRNA ANALYSIS AND TRANSCRIPTOME - Coggle Diagram
mRNA ANALYSIS AND TRANSCRIPTOME
NORTHERN BLOT
We can see the presence of a particular RNA within a sample and also the quantity of the RNA.
Technique not suitable and needs a huge amount of RNA.
Gene specific.
Extraction of total RNA. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly-A tail.
The quality and quantity of RNA can be measured on the gel prior to blotting, then we have to run an agarose gel (polyacrylamide if mRNA) in denaturation conditions to separate size
The RNA needs to be transferred on a membrane to be labelled since the gels are fragile and the probes are unable to enter the matrix. A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them.
The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need of high temperatures, which could cause RNA degradation.
Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane UV light.
We have to amplify by PCR the region we want the probe to detect. The probes are nuclear-acidic complementary to the RNA of interest
Some radiolabelled nucleotide has to be insert to obtain the probe and see it.
The membrane has to be washed carefully then to prevent the background effect. After we can reuse it.
Now we can detect the presence/absence and eventually the quantity of our RNA.
We can use this technique to identify genes linked to:
developmental process
,
metabolic pathways
,
stress
or
drug
.
Compared to RT-PCR, northern blotting has a
low sensitivity
, but it also has a
high specificity
.
Reverse northern blot
In this case the substrate nucleic acid (that is fixed to the membrane) is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled.
IN SITU HYBRIDIZATION
Can provide cellular (and sub-cellular) resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression.
Time-wasting technique.
Gene specific.
In vivo assay.
Whole-mount and RT-PCR
based protocols: increase throughput, but can compromise specificity and resolution. Tissues to be analysed have been undergone to Imbedding in paraffin or polymer; then a labelled RNA antisense probe it's used on sample slices obtained with a microtome.
Whole-mount ISH (WISH)
: using of cell sorting to isolate pure populations of a given cell type from the Arabidopsis root.
Approach limited to species where suitable and diverse cell line markers are available and limited also to tissues whose cells can be separated and sorted
New WISH protocol
: the material is still fixed to prevent RNA degradation and then we do experiment on the whole sample. Data are then analysed by microscope.
Prerequisites to achieve genome-wide coverage in any species
Sequences representing the expressed genes must be suitable for making probes and be available in an organized format.
The remaining manual steps must be further streamlined and, ideally, automated.
Probes
RNA probes
: very stable, tissue can be digested with RNase after the hybridization reducing the background; have a higher specific activity compared to oligonucleotides and they are strand-specific compared to dsDNA probes
Oligonucleotide probes
: better tissue penetration and more specific
Radioactive probes
: not used a lot anymore.
Label
Direct
: use of nucleotides containing a fluorophore
Indirect
: chemical coupling of a modified reporter molecule that can bind with high affinity to another ligand like biotin or digoxigenin
Detection of the marker
Fluorimetric assay
Enzyme assay
WHOLE TRANSCRIPTOME SHOTGUN SEQUENCING (WTSS)
It enables library construction from specific tissue/cells
Uses NGS to reveal the presence and quantity of RNA in a biological sample at a given moment in time.
It is used to analyse the continually changing cellular transcriptome. Facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression
It can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries.
DIFFERENTIAL DISPLAY (DDRT-PCR)
Allows to compare and identify changes in gene expression at the mRNA level between two or more eukaryotic cell samples
All the RNA in each sample is reverse transcribed using a set of 3'
anchored primers
(having a short sequence of deoxy-thymidine nucleotides at the end) to create a cDNA library for each sample.
PCR amplification using arbitrary 5' primers together with anchored 3' primers identical to those used to create the library; about 30 arbitrary primers can amplify almost all the mRNA.
The resulting transcripts are then separated by electrophoresis and visualized, so that they can be compared.
SAGE
Produce a snapshot of the mRNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts
Short nucleotides sequence (no more than 9bp) are able to distinguish 262 thousand transcripts. this technique is based on the creation of huge amount of tag of 9 bp ligated together and then sequenced.
We can discover genes not yet characterized
Output
: list of short sequence tags and the number of times it is observed; statistical methods can be applied to tag and count lists from different samples, in order to determine which genes are more highly expressed -> example of normal tissue and the corresponding tumour
The mRnA of an input sample is isolated and a reverse transcriptase and biotinylated primers are used to synthesize cDNA.
The cDNA is bound to streptavidin beads via interaction with the biotin attached to the primers, and is then cleaved using a restriction nuclease (
anchoring enzyme AE
)
After, the cleaved cDNA downstream from the cleavage site is then discarded.
We have to divide the template (the remaining immobile cDNA fragments upstream from cleavage sites) into two groups: one linked to a specific
adaptor A
, and the other to an
adaptor B
Containing several components in the following order:
Sticky ends with the AE cut site
to allow for attachment to cleaved cDNA;
recognition site for a restriction endonuclease (
tagging enzyme TE
)
;
short primer sequence unique to either adapter A or B
After ligation with the adaptors, the cDNA is cleaved with the TE, we then get the adaptor sequence and the tag of our library.
Link the tags coming from the procedure with the adaptor A with the tag coming from the procedure B.
A and B can be used to do a PCR amplification
After PCR, we have to use the AE to produce a
DITAG
sequence with two different target.
This DITAG is ligated together to form a concatemer to clone to be able to sequence different ligated tags.
We have to clone this concatemer into a plasmid to produce a large amount of sequence enough to be sequenced.
super-SAGE
: use different type of enzymes that can produce tags of 26 bp instead of 9 bp -> longer are the tags sequence and better is the result
miRNA CLONING
Small RNAs are isolated
Linkers are added of each
RNA is converted in cDNA by RT-PCR
The linkers, containing internal restriction site, are digested and the sticky ends are ligated together into concatemers
After concatenation, the fragments are ligated into plasmids and used to transform bacteria to generate many copies of the plasmid containing the inserts
Those may then be sequenced to identify the miRNA present
RT-PCR
The most used method to analyze gene expression
Thermocycle
,
detector of fluorescence
and
laser source
No need to run the gel to analyze the data
It calculates the abundance of RNA so the gene expression in different tissues, developmental stages and organisms
We produce a cDNA with the use of a
reverse transcriptase
The target DNA double during each cycle of PCR. We do at least 30 cycles
PCR is not always 100% efficient: we have to correct the result by the
E corrective parameter
related to the efficiency of the PCR, which depends on the primers constructed
Exponential phase
: particular phase in the PCR where we can be quantitative, after 30 cycles it's no more exponential, so we can't calculate the quantity of the product.
There is a
fluorescence detection
proportional to DNA producing
We can collect the
Ct value
for each reaction that gives the idea of the amount of product in the reaction
We have to construct a
standard curve
with information of the efficiency of the reaction: to build it we have to run different experiments in which we go through a diluition series, we get a linear graph and then we calculate the efficiency, that at least should be
90-105%
Sybr Green
is a non-specific dye that can bind dsDNA, produces fluorescence only if it is bound; it can bind unspecifically
Molecular Beacon Probe
: the fluorescence is proportional to the quantity of the product, it is specific and give a lower background fluorescence.
Taqman probes
: it binds the quencher and the reporter, so the reporter is not able to give fluorescence. As the taq start to produce the template, it is able to degrade the probe and release the reporter that now produce fluorescence. This probe can detect SNPs
Quantification
:
absolute
comparing the Ct value to a standard curve;
relative
done by the ratio between the relative amount of the gene of interest and the amount of a housekeeping gene chosen specifically for the experiment
MICROARRAY TECHNOLOGY
Used in analysing transcriptome of mutants and transgenic plants.
Hybridization based analysis.
The amount of mRNA for each gene in a given sample is measured in parallel with high-throughput into genomic scale
Parameters that influence the hybridization:
sequence composition
(because of the different number of H bonds between AT and GC);
target and probe concentration
;
salt
(high salt = low stringency; low salt = high stringency = more specific);
temperature
(high T = more stringent = more specific; low T = less stringent)
Methods to do microarrays
Printing
Photolithographic
: made by a light source
Types of arrays:
spotted arrays on glass
,
in-situ synthesized arrays
and
self-assembled arrays
Spotted microarrays
: the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs.
Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants by synthesizing this sequence directly onto the array surface instead of depositing intact sequences.
Longer sequence
: more specific to individual target genes;
shorter sequence
: may be spotted in higher density across the array and it is cheaper
Affymetrix
: photolithographic technique to produce oligonucleotide arrays, on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array
Advantages of oligonucleotide arrays
: detect individual gene transcripts, distinguish splice variants, distinguish sense and anti-sense transcripts, the probe redundancy/mismatch strategy helps identify and minimize the effects of non-specific hybridization and background signal, allows the direct subtraction of cross-hybridization signals
Disadvantages of oligonucleotide arrays
: need access to specialist equipment, cannot readily fabricate custom arrays, expensive
Steps to calculate data
Quantifying data
Data pre-processing
Background correction
Normalization
Thresholding
Source of variability
:
Biologic
variability and
technical
variability
Due to the
array fabrication
,
target preparation
,
target hybridization
,
imaging
,
image analysis
,
image quantification
Limitations
: a significant amount of poly A+ RNA is needed and say only information about transcript levels. Expensive.
We confirm microarray data by
RT-PCR
TILING
DNA sequencing method based on hybridization of a universal panel of tiling probes
Millions of shotgun fragments are amplified in situ and subjected to sequential hybridization with short fluorescent probes
High resolution and high sensitivity that, so even small and rare molecules can be detected
miniSEQ
Solid-phase method for the detection of any known point mutation or allelic variation of the DNA
Amplified and biotinylated DNA sequences containing the mutation site are immobilized onto streptavidin coated microplate wells
Primer extension reactions are carried out using labelled nucleotides
Incorporation of the labelled nucleotides is genotype-dependent and it is analyzed using ELISA technique
A DNA sequence containing the mutation site is amplified using 5'-biotinylated primer in a PCR reaction and the product is bound to 2 streptavidin coated microwells.
The bound targets are rendered single-stranded by short alkali treatment and the unbiotinylated strands are washed away
After washing, the solid-phase mini-sequencing reactions are performed in the same wells
The detection primer anneals to the target DNA immediately adjacent to the site of the mutation
It is elongated in a DNA polymerase reaction with a digoxigenin labelled deoxynucleotide corresponding to the nucleotide at the site of the mutation
The DNA polymerase only adds the correctly paired nucleotide onto the 3' end of the detection primer
Since only one nucleotide, dUTP in one well and dATP in the other well, is provided in the extension reaction, the identity of the first nucleotide in the template DNA can be discerned
The wells are washed using using a microplate washer
An enzyme labelled anti-digoxigenin antibody is added and after washing the signals are generated by preferred substrate
AFLP BASED TRANSCRIPT IMAGING
Based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA
cDNA-AFLP
: involves reverse transcription of mRNA into double-stranded cDNA, followed by restriction digestion, ligation of specific adapters and fractionation of this mixture of cDNA fragments into smaller subsets by selective PCR amplification
The resulting cDNA-AFLP fragments are separated on high-resolution gels
Advantage
: no prior sequence information is needed; DNA chips and microarrays; low start-up costs; high specificity
Limitations
: identification of interesting differentially expressed genes requires purifying resulting TDFs from gels followed by amplification and subsequent (cloning and) sequencing; time consuming; labour intensive, not very amenable for automation; when the tags are of insufficient length to characterize the interesting transcript functionally, identification of the corresponding full-length cDNAs might be required