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markers xxx > Week 3 Part 2 > Flashcards

Week 3 Part 2 Flashcards

1
Q

Define transcriptomic biomarker?

A

A measurable RNA characteristic that is an indicator of normal biological process, pathological process, and/or response to therapeutic or other intervention

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2
Q

How much does MRNA occupy?

A

4% of total RNA

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3
Q

What is difference between RNA and DNA?

A
  1. RNA is a single-stranded nucleic acid
  2. RNA has a ribose sugar
  3. RNA nucleotide have uracil base instead of thymine
  4. RNA strands are shorter than DNA strands
  5. AU-CG
  6. RNA forms in the nucleolus and then moves to specialised regions of the cytoplasm depending on the type of RNA formed
  7. RNA is more resistant to damage from UV light than DNA
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4
Q

What is the function of RNA?

A

Convert the genetic information contained within DNA to a format used to build proteins and then move it to ribosomal protein factories

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5
Q

What are the types of RNA ?

A

RNA can be classified into coding RNA and non-coding RNA

  1. mRNA - encoded amino acid sequence of a polypeptide
  2. tRNA - brings amino acid to ribosome during translation
  3. rRNA - the organelles that translates the mRNA
  4. snRNA - form complexes that are used in RNA processing in eukaryotes ( not found in prokaryotes )
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6
Q

What is a non-coding RNA?

A

RNA molecule that is not translated into protein

Examples:

  • tRNA
  • rRNa
  • small RNA: microRNA, siRNA, snRNA
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7
Q

Small nuclear RNA (snRNA)

A

U1, U2, U4-U6 RNA

Splicing if introns

From primary genomic transcripts

Distribution: Eukaryotes and archaea

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8
Q

Small nucleolar RNA (snoRNA)

A

E.g. C/D box snoRNA
H/ACA box RNA

Important for RNA processing

By pairing with proteins to form small nucleolar ribonucleoprotein

Nucleotide modification of RNAs

Distribution: Eukaryotes and archaea

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9
Q

What inhibits gene expression?

A

MicroRNA and siRNA

SiRNA - gene regulation

Distribution: most eukaryotes

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10
Q

What is used for gene silencing?

A

A cell can use MicroRNA

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11
Q

What is the function of MicroRNA?

A

Turn off genes by inactivating messenger RNA which are responsible for translating the genetic information into proteins

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12
Q

What does MicroRNA participate in?

A

Regulation of the cell from its development to its death

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13
Q

What serious consequence can dysregulstion of MicroRNA have?

A

For our body

Cause a range of diseases such as cancer and heart disease

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14
Q

What is the microRNA biogenesis and function?

A
  1. The gene is contained in the nucleus in the DNA
  2. Each gene is transcribed by RNA polymerase 2 which either produces a regulatory or messenger RNA
  3. The transcript is a primary MicroRNA which form a typical hairpin loop structure - becomes final MicroRNA with regulatory function after several steps of processing
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15
Q

What is the process of biogenesis and function of MicroRNA?

A
  1. The double-stranded stem is recognised by a protein DGCR8
  2. An enzyme DROSHA associates DGCR8 to form a micro processor complex which is able to cut RNA into smaller precursor MicroRNA - imported into cytoplasm where it will inactivate messenger RNA of one or multiple genes
  3. The precursor MicroRNA is carried out of the nucleus through the nuclear pore by the transporter molecule Exportin 5
  4. In the cytoplasm it is recognised by a large RNAse protein called DICER
  5. DICER cleaves the stem loop and forms the short double-stranded MicroRNA molecule
  6. Protein AGO-2 interacts with DICER to bind MicroRNA
  7. The MicroRNA is unwound and one strand is released
  8. The remaining strand interact with AGO-2 and additional proteins to form the RISC
  9. Guided to its target and inactivate one or multiple genes
  10. Messenger RNA is a target gene is complementary to the sequence of MicroRNA that enables space pairing
  11. Once bound there are 2 ways in which RISC can inactivate the mRNA
  12. Proteins and the complex can simply cut the messenger RNA which while be further destroyed by cell
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16
Q

What is RISC complex involved in?

A
  1. Metabolism
  2. Stem cell division
  3. Development
  4. Apoptosis
  5. Cell proliferation
  6. Cell cycle control
  7. Stem cell differentiation
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17
Q

How many miRNA does the human genome have and what are their function?

A

1,048 miRNA

Regulate virtually all biological processes

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18
Q

What does miRNA execute?

A

Posttranscriptional gene silencing through mRNA destabilisation as well as translational repression

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19
Q

What do miRNA form base pairs with?

A

Specific sequences in protein-coding mRNAs

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20
Q

What does near-perfect pairing induce?

A

Cleavage of target mRNA

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21
Q

What does partial pairing result in?

A

Translational repression

mRNA decay through de-adenylation pathways

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22
Q

What may miRNA regulate?

A

More than 1/3 of all protein-coding genes and virtually all biological processes

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23
Q

What does the sources of RNA have a role in?

A

Regulatory role in every organism

And in every biological function influencing normal biology and disease process

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24
Q

Where is most of miRNA knowledge coming from?

A

Cancer research

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25
Q

Where are extracellular miRNA present?

A

In many other biological fluid including:

  1. Saliva
  2. Tears
  3. Seminal fluid
  4. Breast milk
  5. Vitreous and aqueous humours of the eye
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26
Q

What is urine a source of?

A

RNA biomarker for urological cancers

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27
Q

What is the potential role of cerebral spinal fluid?

A

MiRNA in brain tumours

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28
Q

What has milk recently been identified as?

A

Potential supply of RNA biomarkers in breast cancer patients

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29
Q

the aqueous humour of eyes appear to be what?

A

Promising source of extracellular miRNA for diagnosing glaucoma

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30
Q

What can cells export?

A

RNA packages in 30-150nm vesicles called exosomes

Taken up and translated by recipient cells - intercellular communication

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31
Q

What are examples of membrane enclosed vesicular bodies?

A
  1. Nanovesicles
  2. Shedding vesicles
  3. Microvesicles
  4. Microparticles
32
Q

What is involved in neuronal function such as pre- and post-synaptic retrograde signalling?

A

Exosomal and extracellular vesicles

33
Q

Where can extracellular RNA (mainly miRNA) be found?

A

Stably outside vesicles in complex with lipoproteins such as:

  1. HDL
  2. Argonaute2
34
Q

What is the origin of majority of extracellular RNA circulating in plasma/serum?

A
  1. Haematologic

2. Endothelial cell origin

35
Q

What is the majority of extracellular RNs circulating in CSF?

A

Derived from:

  1. Oligodendrocytes
  2. Microglia
  3. Macrophages
  4. Neurons
36
Q

How can high quality RNA and pure RNA be isolated?

A
  1. Commercial kits based on a combination of a lysis step and column precipitation
37
Q

Where can RNA be extracted from?

A

Dried blood spots which has been stored at room temperature for a few years

38
Q

What can profiling RNA expression pattern facilitate?

A

Presymptomatic disease detection

39
Q

What do living neurons and other CNS secrete?

A

MicroRNA and other small non-coding RNs into extracellular space packaged in exosomes, micro vesicles or lipoprotein complexes

40
Q

What are the sources for representative RNA?

A
  1. Body fluids
    - blood
    - plasma
    - CSF
  2. Non-neuronal tissue
  3. Cells such as lymphocyte
41
Q

What are the number of different methods to detect small RNAs (MicroRNA)?

A
  1. Northern blot
  2. In situ hybridisation
  3. QRT-PCR
  4. Microarray
  5. Small RNA sequencing
  6. Solid-based technologies
42
Q

What are the advantages of solution-based techniques?

A
  1. Much faster output
  2. Can be used in vivo
  3. Miss the global picture
43
Q

What are the several steps RNA-seq data analysis?

A
  1. Quality control
  2. Alignment to reference genome
  3. Read quantification/ count to determine the expression levels
  4. Statistical comparison between the groups (large datasets require p values corrected for multiple testing)
44
Q

For biomarker research involving large datasets, what is the common approach?

A
  1. Unsupervised (no prior knowledge of sample grouping) approaches
    - hierarchical clustering (e.g. heatmap)
    - principle component analysis (PCA)
45
Q

What does blood-based biomarker test use and and what doesn’t it allow evaluation of?

A
  1. Use plasma or serum

2. Doesn’t allow evaluation of both intracellular and extracellular changes

46
Q

What is the method for MicroRNA in new born with brain injury?

A
  1. Extract and compare expression of miRNA from single small 6-mm- diameter dried blood spot stored at room temperature with those from EDTA - blood, plasma and urine
  2. Three miRNA (RNU6B, let7b, miR21) were quantified via extraction and quantitative RT-PCR performed from DBS - compared with EDTA
47
Q

What was evaluated as potential biomarkers for HIE?

A

Candidate miRNA:

  1. Let7b
  2. MiR21
  3. Mir29
  4. Mir124
  5. Mir155

In DBS

48
Q

What was the results of biomarkers in newborn with brain injury?

A
  1. Candidate miRNA were extracts me in all biosamples from new-born
    - highest expression in DBS
  2. Good correlation between miRNA level in DBS and EDTA blood at -80 degrees
  3. No significant difference was observed in the miRNA levels between the favourable and unfavourable outcome groups for babies with HIE
49
Q

What is the conclusion for biomarkers with babies that have brain injury?

A
  1. DBS May be useful for studying the potential of miRNA as biomarkers for brain injury
50
Q

Where was RNU6B miRNA not detectable?

A
  1. 4/30 plasma sample

2. 8/30 urine sample

51
Q

What was correlation carried out for?

A
  1. Expression of RNU6B, let7b and miR21 in all biofluids with DBS
  2. Positive correlation
52
Q

No statistically significant correlation between

A
  1. DBS with plasma or urine
53
Q

What was still possible to extract after

Prolonged storage?

A
  1. Selected miRNAs from all the samples
54
Q

What did Taqman MicroRNA use?

A

10 ng total RNA

55
Q

Where was the level of MicroRNA greatest?

A
  1. DBS compared to other biofluids
56
Q

What did Hierarchial clustering of RNA-seq data show?

A
  1. Some cluster
    - especially cord blood from healthy newborns compared with newborns with brain injury

Next step: identify the MicroRNA and validate in an independent cohort of newborns with brain injury

57
Q

MicroRNA in Parkinson’s disease (intro)

A
  1. PD is a devastating neurodegenerative disorder
    - preferential loss of dopaminergic (DA) neurons in substantia nigra pars compares (SNpc)
    - presence of intracytoplasmic inclusions (Leah bodies) + lewy neutrites in neurons
58
Q

What causes an autosomal-dominant form of familial PD?

A
  1. Multiple missense mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene
59
Q

What does recent GWAS reveal?

A
  1. LRRK2 gene locus is a genetic risk factor for the more common sporadic PD
  2. Indicate a potential alteration in the LRRK2 expression in aetiology of sporadic PD
60
Q

What has a number of miRNA been associated with?

A
  1. Neuronal development
  2. Synaptic plasticity
  3. Memory formation
  4. Neurodegenerative diseases in the nervous system regulating the translation of targeted genes
61
Q

What is the function of miR-133b?

A
  1. Indicated in the development of midbrain DA neuron
62
Q

What does Mir-7 repress?

A
  1. The expression of alpha-synuclein protein

2. Protects against alpha synuclein-mediated cytotoxicity

63
Q

What is LRRK2 actively involved in?

A
  1. miRNA processing
64
Q

What was found when level of LRRK2 protein and LRRK2-targeting miRNA In the frontal cerebral cortex of patients with sporadic PD were examined?

A
  1. Level of LRRK2 protein expression was significantly increased in PD brains
  2. Level of miRNA-205 expression was decreased
65
Q

What is the function of Mir-205?

A
  1. Provide a therapeutic avenue to inhibit LRRK2 expression in the sporadic PD
66
Q

MicroRNA in Parkinson’s disease (methods)?

A
  1. Subjects of legal representative signed informed consent approved by ethics review board
  2. rapidly autopsied frontal cerebral cortex were obtained from brain bank
    - sporadic PD (n=8)
    - sporadic PD with dementia (n=8)
    - non-pathological controls (n=7)
  3. Confirmation of PD was based on clinical features and Lewy bodies within pigemented neurons lost in substantia nigra (dopaminergic neurons)
  4. Total RNA were prepared from brain tissue using RNA isolation kit
  5. The level of LRRK2 mRNA expression were normalised with actin mRNA
67
Q

MicroRNA in Parkinson’s disease (results)

A
  1. Expression of LRRK2 protein in the frontal cortex of PD and PDD patients were significantly unregulated compared with NPC [non-pathological control]
68
Q

MicroRNA in Parkinson’s disease (discussion)

A
  1. Conserved Mir-205 binding site on LRRK2 gene across different vertebrate species was identified
  2. Mir-205 can modulate expression of low-density lipoprotein receptor-related protein 1(LRP1)
  3. Although the frontal cortex in PD show no cell death like substantia nigra pars compacta, it is still molecularly and pathologically affected in PD
  4. It remains unclear how Mir-205 expression is down-regulated in brains of PD
  5. Not useful in living patients
69
Q

MicroRNA in Parkinson’s disease (CSF study method)

A
  1. Written informed patient consent
  2. CSF samples from Parkinson’s disease (n=28)
    - Multiple System atrophy (n=17)
    - Non neurological controls (N=30)
  3. CSF samples criteria:
    - leukocyte count <5 cells
    - erythrocyte count < 200 cells
    - about blood contamination as blood cells in CSF affect miRNA levels
  4. CSF samples collected in tubes, centrifuged, aliquoted and stored at -80 degrees
  5. RNA isolation, reverse transcription into cDNA, pre-amplification and qPCR
  6. Three samples were used as inter-plate control for qPCR reactions to determine inter-plate variation in miRNA quantification
  7. Selection of miRNA targets was based on previously publications with potential as a biomarker for PD or MSA
70
Q

MicroRNA in Parkinson’s disease (serum study method)

A
  1. Study was conducted in accordance with Helsinki Declaration
  2. Fasting venous blood samples (4ml) were collected via vein puncture using coagulation-promoting tubes
  3. Blood was left to stand 30 mins at 4 degrees Celsius
    - centrifuged at 2000g for 15 mins
    - serum stored at -80 degrees
  4. Exosomes were isolated using a commercial total exosome isolation kit
  5. Synthetic cel-mir-39 (40pM) was added to exomsome supernatant to control and normalise RNA extraction efficiency before total RNA extraction
  6. Isolated RNA was quantified using qPCR
71
Q

MicroRNA in Parkinson’s disease (CSF and serum studies results)

A
  1. Mir-205 was shown to be increased in PD CSF vs control, but was not studied in serum
  2. Mir-24 was shown to be decreased in CSF vs controls and increased in serum vs control
  3. Mir-19b was shown to be decreased in CSF for MSA, but not PD, and decreased in PD serum vs control
  4. Other MicroRNAs tested were not significant
72
Q

MicroRNA in Parkinson’s disease (discussion)

A
  1. Mir-205 was shown to be decreased in post-mitten brain tissue and increased in CSF vs control
  2. Mir-24 was shown to be decreased in CSF vs control and increased in serum vs control
  3. Mir-19b was shown to be decreased in MSA, CSF but not PD and decrease in PD Vs control
73
Q

What are limitations in biomarker research?

A
  1. Validation

2. Standardisation

74
Q

Validation

A
  1. Often first pilot study carried out in small sample size
  2. Usually validate in a small same size as well
  3. Does not replicate previous finding
  4. Need to use large and independent cohort to confirm robustness and reproducibility of biomarker candidate
75
Q

Standardisation

A
  1. Handling of samples and carrying out the molecular assay
  2. Need to have agreed standard operating procedures and good laboratory practice to ministry variability between different studies
76
Q

What are advantages of MicroRNA as a biomarker in neuroscience?

A
  1. Present in a variety of biofluids (e.gx blood, urine, saliva)
  2. Stable in a variety of biofluids and resistant to enzyme digestion (e.g. resistant to RNAse present in blood)
  3. Withstand extreme temperatures, PH, FFPE process, and multiple freeze-thaw cycles
  4. Easily detectable using various lab techniques
  5. Respond to many CNS disease
  6. Potential ad effective biomarker for neuroscience disease

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