RNA Flashcards
What is Crick’s Central dogma?
A unidirectional 1:1 commentary on the flow of genetic information:
DNA - RNA - Protein - Function
Briefly outline factors which have resulted in a revision of Crick’s central dogma
One piece of DNA can become several different RNAs due to alternate splicing
Different functional proteins can. be produced from each RNAs due to post-translational modification
There can be areas of reverse transcription (RNA - DNA) mean the flow of information is not always unidirectional
MicroRNA can also inhibit the flow of information
Epigenetic can affect the transcription of DNA
The transcriptome is fixed. T/F?
False
What are the various subtypes of RNA?
mRNA
tRNA
rRNA
miRNA
Briefly describe the structure of RNA
RNA is ribonucleic acid. It has a ribose-phosphate backbone and has a uracil in place of thymine. Its single stranded structure makes it less stable than DNA. It has no introns
When working with RNA, why is it important to use RNase ZAP on all surfaces?
Because RNA is readily degraded by a variety of mechanisms (thermal, chemical, enzymatic), so good preparation of samples and workspaces is vital
Working with RNA quite often involves working with small volumes. T/F?
True
Describe how RNA can be stabilised and isolated for use in experiments.
Sample RNA need to be protected from degradation by snap freezing - liquid nitrogen or RNA later
Extraction occurs by homogenising the tissue/cells which are usually in the frozen state
Then a series of steps is used to neutralise RNAses, shear genomic DNA, bind RNAs to specific membranes, remove. contaminants and elute isolated RNA - this can now all be done using off-the-shelf kits which generate good yields of high quality RNA rapidly
When producing an RNA sample for experimentation, how is RNA quantified?
Quantification is by measuring the sample’s absorption of light. The 260:280 wavelength ratio is an important indicator of purity. This is the ratio of absorption of light of 260nm compared to 280nm. Nucleic acids will absorb light of 260nm whilst proteins will absorb light of 280nm. Thus the spectrophotometer can measure purity.
Describe an alternative method to spectrophotometry that can be used to assess the quantity of RNA produced when creating an RNA sample
Use of a fluoresent tag that binds to nucleic acid and selective fluorescence when bound. The intensity of the fluorescent dye can then be measured
What are the advantages and disadvantages of using fluorescent tags to spectrophotemetry to quantify. RNA?
Useful when concentration too low for spectrophotometry to provide an accurate assessment
More sensitive - can. be used to detect presence of contaminants absorbing at 260nm
Higher price per sample
Lengthier sample preparation process
Why are RNA samples synthesised back into (c)DNA for experiments?
DNA is more stable and therefore easier to work with as it is double stranded
DNA can be used in various downstream PCR and other molecular biological techniques
Describe the materials required for 1st strand cDNA synthesis
Template of isolated RNA Reverse transcriptase enzyme Buffers dNTPs primers
Describe the different primers that may be used in cDNA synthesis?
Gene specific primers - amplify only one gene of interest
Oligo-dT primers - made of a string of thymines that bind to the string of adenosines on the tail of mRNA,
Random hexameters - primers of 6 basepair fragments that. can anneal along the RNA sequence at any. point at which their sequence is complementary so can be used to amplify. degraded RNA without a tail
Describe scenarios in which oligo-dT primers would be the primer of choice?
Constructing cDNA libraries from eukaryotic mRNAs
Full-length cDNA cloning
3’ rapid amplification of cDNA ends
Describe scenarios in which oligo-dT primers would not be appropriate?
Not suitable for degraded RNA
Not suitable for RNAs that lack poly(A) tails such as prokaryotic and microRNAs
Can cause 3’ bias - RNA with significant secondary structure may also disrupt full-length cDNA synthesis, resulting in under representation of the 5’ ends
Describe scenarios in which random (hexamer) primers would be the primer of choice?
Production of cDNA from RNAs without poly(A) tails (rRNA, tRNA, non-coding RNAs, small RNAS, prokaryotic mRNA), degraded RNA and RNA with known secondary structures
Why is it important to start with. equal amounts of material in each sample when producing cDNA?
Given that cDNA is a hybrid molecule (contains one strand fo DNA and one of RNA) there is no gold standard way to accurately quantify cDNA
There is also an assumption that the reverse transcription process. has equal efficiency across all of the. RNAs present.
What is reverse transcription PCR?
In PT-PCR an RNA population is converted to cDNA by reverse transcription and then the cDNA is amplified by PCR
What are the applications of RT-PCR?
Detection of expressed genes
Examination of transcript variants
Generation of cDNA templates for cloning and sequencing
In one-step RT-PCR the production and amplification of cDNA occur in the same reaction. tube. What are the advantages and disadvantages of this?
Advantages:
Simplifies workflow, reduces variation, minimises possible contamination
Disadvantages:
Uses gene-specific primers so limits analysis to few genes per sample, could. be less sensitive and less efficient
What is a cDNA library?
A cDNA library consists of cDNA clones that represent the transcribed sequences within a specific sample
What are the applications of cDNA libraries?
Identification of novel genes, identification of splice variants and generation of templates for full length cDNA analysis.
Why is screening of a cDNA library less resource intensive than screening an equivalent genome DNA library?
Because cDNA contains no introns or untranslated regions so is much smaller
How do gene expression microarrays. differ from DNA microarrays?
DNA microarrays use genomic DNA as a template, gene expression microarrays use cDNA as a template
Gene expression microarrays is less sensitive than qRT-PCR. How is this problem overcome?
Usually a microarray is used to identify targets and these are then validated using taw-man/
Typically, microarray experiments generates vast quantities of data. Give an example of a technique which helps us handle this data?
Ingenuity pathway analysis
Describe the steps in the process of northern blotting
The first step is to denature the RNA and then separated the strands by size using gel electrophoresis.
The RNA molecules are then transferred form the gel onto a blotting membrane.
Next, the membrane is treated with a small piece of DNA or RNA called a probe, which has been designed to have a sequence complementary to a particular RNA sequence of interest in the sample. This allows the probe to hybridiser bind to a specific RNA .
The probe has a radioactive or fluorescent label that permits its detection.
What are the applications and advantages of northern blots?
Determination of bio distribution of an expressed gene
Determination of the size of a transcript
Detects presence of split variation or other processing of the mRNA
Estimation of expression levels
Good degree of sensitivity, high specificity
Membrane stable over months to years
What is in-situ hybridisation
A type of hybridisation that uses a labelled cDNA, RNA or modification nucleic acid strand probe to localise a specific RNA sequence in a portion or section of tissue (in situ)
What are the challenges associated with in-situ hybridisation?
Requires preservation of target mRNA within tissue which can be difficult
Requires very thin tissue sections
Common methods of preparing samples include freezing which can result in freeze artifacts occurring.
What is the gold standard method of accurately quantifying the amount of a ds-nucleic acid?
qRT-PCR
Describe the process of qRT-PCR
cDNA material is denatured, a primer and probe (with reporter and quencher molecule) are attached
Normally, the quencher molecule prevents fluorescence but when tan polymerase reaches the probe, it liberates. the reporter molecule so that fluorescence occurs. Changes in the fluorescence curves indicates changes in the amount of the gene of interest in the starting material.
In qRT-PCR, what does a high Ct value indicate?
The higher. the Ct value, the more cycles were taken to reach a certain level. of fluorescence (threshold point), therefore, the less of the. template there was to begin with.
Explain the difference between absolute and relative quantification in qRT-PCR?
Absolute quantification - analysis is performed in conjunction with. a standard curve consisting of serial dilutions of a source (e.g. plasmid) of the gene/cDNA of interest
Relative quantification - quantification is relative to a specific (usual control) group
What is a housekeeping gene?
They are typically constitutive genes that are required for the maintenance of basal cellular functions that are essential for the existence of. a cell, regardless of its specific differentiated role. They are expressed in a ll cells of an organism under normal and pathology-physiological conditions, irrespective of tissue type, developmental stage, cell cycle state or external signals.
Why are housekeeping genes used in qRT-PCR?
They act as an internal control as these genes will be expressed in all cells
What are miRNAs?
Short non-coding RNA molecules that act as negative regulators of gene expression by inhibiting RNA translation or promoting mRNA degradation
miRNAs can only target a single mRNA molecule. T/F?
False - a single miRNA can. target from a few to many. individual mRNA molecules
Describe the production of miRNA and how it carries out its role
miRNA can sit within genes or intron. Formation starts with pro-miRNA then pre-miRNA, then in the cytoplasm they are modified and become incorporated in the. RISC complex. When inside the RISC it can mark mRNA for translational repression or degradation
What are antagonisers?
Genetic engineered oligonucleotides that prevent other molecules from binding to a desired site on an mRNA molecule - used to silence endogenous microRNA
How can miRNAs be targeted therapeutically?
By inhibiting them to prevent them from inhibiting RNA expression or by mimicking them to turn off gene expression
What are the advantages of therapeutically exploiting miRNAs?
miRNAs can regulate multiple components of the same pathway/cellular process
Long lived sustained effects
Target mRNA is defined
Effective in-vivo regulation of mRNA
What are the disadvantages of therapeutically exploiting miRNAs?
Challenge in delivery of miRNA modulator
Single miRNA can have both beneficial and pathogenic effects
Toxicity of miRNA modulator
How do miRNAs result in silencing of mRNA molecules?
Cleavage of the mRNA strand into two pieces
Destabilisation of the mRNA through shortening of its poly(A) tail
Less efficient translation of the mRNA into proteins by ribosomes
What is the term for miRNAs which are associated with cancer?
Oncomirs
