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mRNA ANALYSIS AND TRANSCRIPTOME, How to quantify the products ? - Coggle…
mRNA ANALYSIS AND TRANSCRIPTOME
why study the transcriptome?
we want to see the comeplete set of mRNA of cells, tissue or organs to detect the differential ones
important because
the transcriptome correlates with developmental steps, metabolic pathways, stress, drugs and other conditions
**HOW TO STUDY THE TRANSCRIPTOME
NORTHERN BLOT
PROCESS
➡︎ we start with the
extraction of total RNA
.
➡︎
The quality and quantity of RNA can be measured on the gel prior to blotting
➡︎ RNA is then transferred on a memebrane to be labelled since the gels are fragile and the probes are unable to enter the matrix (they cannot pass though the agarose gel)
For northern blotting a nylon membrance with positive charge is used and the transfer buffer used for the blotting contains
formamide
that lowers the anneling temperature of the probe-RNA interaction. The RNA is immobilized to the membrane though covalent linkage by UV light.
➡︎ we have to amplify by PCR the region we want the probe to detect
➡︎ radiolabelled nucleotides (radioactive isotopes or chemo-luminescence) have to be inserted for the probe to see it → labelling can be done by radioactive isotopes (P32) or by chemoluminescence
➡︎ after a careful washout we can
detect the presence-absence an eventually the quantity of our DNA
☺︎ NB can detect small changes in gene expression that microarrays cannot
☺︎ has high specificity
☹︎ NB can look just at a few genes at the time
☹︎ low sensitivity compared to RT-PCR
simple, cheap, fast and we able to see the
presence of a particular RNA
within a sample and also the
quantity of the RNA
we can use it to **identify genes liked to developmental processes, metabolic pathways, stress drugs
➔ Expression (RNA-level) might tell us something about the gene function
IN SITU HYBRIDIZATION
➡︎
it is a difficult and time wasting technique
➡︎ It is a gene specific technique
➡︎ it is an in vivo assay
➡︎
with the advent of GWAS there is a urgent need to develop high-thoughput in situ methods that can also provide high resolution
whole-mount ISH (WISH)
PROCESS
:
☛ we can use different types of
probe
:
RNA
ones are very
stable
and have a
higher specificity
compared to oligonucleotides and they are
strand specific
compared to dsDNA probes
Oligonucleotides
ones have a
better tissue penetration
➡︎ Probes can be labelled
directly
(use of nucleotides containing fluorophore) or
indirectly
(chemical coupling of a modified reporter molecule that can bind to another ligand, the nucleotide is linked to a reported and is then seen, by and antibody, and detected ➨ the marker can be detected in various ways: either by a fluorimetric assay or it can be coupled to an enzyme assay where it will produce a product and then measured colorimetrically ➧
BIOTINE-STREPTAVIDEINE and DIGOXIGENIN
(REPORTER GROUPS)
we have to choose a super specific region of the target, so we can clone it into a vector and use T7/T3 or SP6 promoters to synthetize the probe in vitro
according to these models, classes of genes are specifically expressed according to a specific pattern
the new protocol retains high resolution and specificity but integrates a degree of automation to a standardized and streamlined protocol
certain prerequisites are necessary:
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
provides cellular and sub-cellular resolution of mRNA levels within multicellular organisms → used to provide
spatial and temporal information on gene expression
➡︎ we can detect the expression of more then one transcript, this can be done if we design different probes differently detectable ( for example with different colour)
cDNA LIBRARY SEQUENCING
makes the sequencing of many clones feasible
enables library construction from specific tissues/cells → and so a database development leading to gene discovery
to analyze cDNA
RNAseq
it can look at different populations of RNA :
a) total RNA
b) small RNA
c) ribosomal profiling
d) exon/intron boundaries
e) verify and amend previously annotated 5' and 3' gene boundaries
facilitates the ability to look at:
a) alternative gene spliced transcripts
b) post-transcriptional modifications
c) gene fusions
d) mutations
e) changes in gene expression
➡︎ used to reveal the presence and quantity of RNA in a biological sample at a given moment in time
➡︎ used to analyze the continually changing cellular transcriptome
DIFFERENTIAL DISPLAY
laboratory technique that allows ro compare and identify changes in gene expression at the mRNA level between two or more eukaryotic samples
then it was surpassed by microarray approaches
SAGE
technique used to produce a
snapshot of the mRNA population in a sample of interest in the form of small tags (9 bp) that correspond to the fragments of those transcripts
➔ the output is a
list of short sequence tags and the number of times it is observed
analyzing this output
1. count the tag in different conditions
2. with sequence databases we can determine from which original mRNA (so which gene) that tag was extracted
3. we can apply statistical methods to determine which genes are more highly expressed
⇝ we can compare a normal tissue and the corresponding tumor
PROCESS :
➤ the
mRNA of an input sample is isolated
➤ a reverse transcriptase and biotinylated primers are used to
synthesize cDNA from RNA
➤ the cDNA is bound to
Streptavidin beads
and it is
cleaved with the AE (anchoring enzyme)
➤ the cleaved cDNA downstrem from the cleavage site is discarded
➤ we divide the template into two groups :
one linked to a specific adaptor A
the other B
⇨ these adaptors have :
sticky ends with the AE cut site
a
recognition site
for a restriction endonuclease (TE)
a
short primer sequence
unique to the adapter A or B
➤ After the ligation with adaptors the cDNA is cleaved using TE enzyme →we get the adaptor sequence and the tag of our library
⇨
we link the tags from the procedure with the adaptor A with the tag coming from procedure B
⇨ PCR amplification
⇨ using the anchoring enzymes we then produce
a DITAG sequence
with two different target and this is then ligated together to form a
concatemer
to clone and be able to sequence different tags
this technique was improved using different enzymes that are able to produce tags of 26bp instead of 9bp ➡︎
SUPER-SAGE
the goal of this technique is similar to the DNA microarray BUT:
based on sequencing mRNA output, not hybridization of mRNA output to probes, so
transcription levels are measured more quantitavely
than microarray
mRNA sequences do not need to be known a priori so we can
discover new gene or gene variants
miRNA CLONING
procedure is similar to SAGE:
⇒ small RNAs are isolated
⇒ linkers are added to each
⇒ the RNA is converted to cDNA by RT-PCR
⇒ the linkers are digested and the sticky ends are ligated to create concatemers
⇒ the fragments are ligated into plasmids and used to transform bacteria to generate many copies of the plasmid contianing the inserts
⇒ those may then be sequenced to identify the
miRNA present
as well as analysing the
expression levels
PCR
RT-PCR
most used method to analyze gene expression
is a technique able to
quantify the amount of product produced in each cycle
→ it calculates the abundance of RNA
PROCESS:
⁍ we start with cDNA produced from the starting RNA in the sample
⁍ we use
REVERSE TRANSCRIPTASE
for the conversion of RNA into cDNA with the Polymerase (RT) + dNTPs
⁍
PCR
➜ procedure in which the target DNA double during each cycle → at least 30 cycles, then it reached a
plateau
, after the 30 cycles the growth is not exponential anymore
➔ The quantity of the PCR is correlated with the initial amount of cDNA
⁍ We have to follow the reaction so we can control if it is still in exponential phase ➔
FLUORESCENCE DETECTION
(proportional to the DNA producing)
⁍We can construct a standard curve with information of efficiency of the reaction
⇨
different labelling methods can be used for quantification
How can we quantify the PCR product?
☞
Sybr green
: is a non specific dye that can bind dsDNA and we need to optimize the reaction since it binds unspecifically
☞
Molecular Beacon Probe
: when the template is available the probe recognize it and binds to it, the fluorescence is proportional to the quantity of product since it is activated by the quencher
☞
Taqman probe
: it binds to the product during the extension phase. At this point the quencher and the reporter are close so the reporter is not able to give fluorescence , but as the taq starts to produce the template is able to degrade the probe and it releases the the reporter that will give the fluorescence
DIGITAL PCR
different from RT-PCR since it gives a
yes-no output (digital)
➔ we split the sample in smaller volumes
➔ we do PCR to see if we have amplification or not
➔ if the transcript is abundant we have many positive, if it is lower only a few and the number of positive reaction depends on the abundancy of the template
➟ we get an absolute quantification
MICROARRAY TECHNOLOGY
it is a
HYBRIDIZATION BASED ANALYSIS
parameters that can influence hybridization:
a) Sequence composition
→ different number of H bonds between AT and GC
b) Target and Probe concentration
c) salt
→ high salt concentration = low stringency and low salt concentration = high stringency
d) temperature
→ high temperature= more stringent (more specific), low temperature = less stringent
3 basic types of arrays come to play :
SPOTTED ARRAYS
➔ The probes are
oligonucleotides
,
cDNA
,
small fragments of PCR products that correspond to mRNAs
➡︎ The probes are synthesized prior to deposition on the array surface and then are "spotted " onto glass
➡︎ A common approach utilizes an array of fine pins or needles dipped into wells containing DNA probes and the depositing each probe at designated locations on the array surface
➡︎ the resulting grid of probes is ready to receive the complementary cDNA or cRNA targets
it is a technique used to produce
"in-house" printed microarrays
since they can also be easily customised for each experiment but
may not be at the same level of sensitivity compared of commercial oligonucleotide
OLIGONUCLEOTIDE MICORARRAYS
oligonucleotide arrays are produced by printing short oligonucleotide sequences to
represent a single gene or family or family of gene splice variants
by synthesizing this sequence directly onto the array surface instead of depositing intact sequences
sequences might be LONGER ➔ more specific to individual target genes
or SHORTER ➔ spotted in higher density and are cheaper to manufacture
it is a
photolithographic synthesis
: light sensitive masking agents are used to build a sequence one nucleotide at the time
Advantages
:
1- detect individual gene transcripts
2- Distinguish splice variants
3- distinguish sense and antisense transcripts
4- the probe redundancy/mismatch ➔ identify and minimize the effect of non specific hybridization and background signal
5- direct subtraction of cross-hybridization signals
Disadvantages
:
1- specific equipment needed
2- cannot readily fabricate custom arrays
3- expensive
IN-SITU SYNTHESIZED ANALYSIS
SELF - ASSEMBLED ARRAYS
two methods to do the microrarrays
Printing
Photolitographic
(light-source)
TILLING
DNA sequencing method based on hybridization of a universal panel of tilling probes
Millions of shotgun fragments are amplified and subjected to sequential hybridization with short fluorescent probes
the sequencing chemistry is simple, enzyme free and consumes only dilute solution of the probes ➔ reduced sequencing cost and increased speed
Solves a lot of problems of traditional methods
MINI-SEQUENCING
fast and simple
amplified, biotinylated DNA sequences containing mutation site are immobilized onto streptavidin coated microplate wells
Primer extension reactions are carried out using labelled nucleotides
incorporation of the labelled nucleotide is analyzed using ELISA
AFLP BASED TRANSCRIPT IMAGING
How to quantify the products ?
absolute quantification
:
comparing the CT value to a standard curve
relative quantification
:
ratio between the relative amount of the gene of interest and the amount of a housekeeping gene chosen specifically for the experiment