Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

7BBFM128: Forensic Biology > Tissue identification, mRNA profiling and DNA methylation > Flashcards

Tissue identification, mRNA profiling and DNA methylation Flashcards

You may prefer our related Brainscape-certified flashcards:
AP® Biology flashcards Biology 101 flashcards
1
Q

LO

A
  • Epigenetics
  • The fundamental differences between DNA and RNA
  • RNA applications in forensics
  • mRNA profiling historical overview and modern advancement
  • DNA methylation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is epigenetics and what are some changes that can affect epigenetics?

A
  • epigenetics is the study if heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in DNA sequence
  • Cells differentiate into different cell type starting from the gamete cells
  • Modification in the chromatin status can affect gene expression and therefore protein expression and cell functioning
  • Effecting the epigenetic changes:
    o Histone post-translational modifications
    o Chromatin looping
    o Non-coding RNAs
    o DNA modification
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

The central dogma

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Compare the molecular structures of DNA and RNA

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What are the different types of RNA and their function?

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Tell me about RNA folding

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Tell me about RNA folding

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Tell me the stages to Messenger RNA or mRNA production

A
  1. Simple chain produced in the nucleus-** Transcription **
  2. G-Cap added to the 5’ end
  3. PolyAtail addition on the 3’ end
  4. Splicing events occur
  5. mRNA leaves the nucleus
  6. Translation in the cytoplasm
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How does miRNA form a loop?

A
  • G-cap and PolyAtail important else degradation occurs if it was single stranded
  • Uses PolyAtail and G-cap to bind together and form a loop which provides protection, this also is where the protein starts building from
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Tell me about the RNA regulation process of translation

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How is miRNA formed from the miRNA gene?

A
  • Modulating the response eg using miRNA
  • miRNA is shorter and don’t need specific genes
  • 20-40 bp: miRNA
  • miRNA transcribed from DNA
  • Drosha and Pasha bind to miRNA and can leave nucleus via gene called exportin 5
  • Froms TRBP/Loqa and dicer loops?
  • Forms RISC-like miRNP comex

Human miRNA biogenesis is a two-step process, with both nuclear and subsequent cytoplasmic cleavage events performed by two ribonuclease III endonucleases, Drosha and Dicer
The miRNA gene is transcribed to produce a primary miRNA (pri-miRNA) that is processed into a precursor miRNA (pre-miRNA) and subsequently miRNA duplex (miRNA:miRNA*, passenger strand designated with asterisk) which ultimately releases mature miRNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Tell me about mRNA target recognition and how this helps with the modulation process?

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

RNA in forensics, how is it used?

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How is RNA used in PMI evaluation?

A
  • In a living cell rRNA and tRNA are stable for days, mRNAs normally has a short half-life
  • When death occurred, cell ribonucleases start degraded RNA and it was believed that the mRNA was destroyed shortly after
  • Recent studies show that isolation of intact RNA is possible several days or weeks post-mortem
  • Beside enzymatic degradation, RNA decay might also be influenced by external factors like sun light, humidity, or high temperatures during the post-mortem interval (PMI)
  • Pancreas and liver have high ribonuclease activity, other tissues such as brain show greater stability up to 96 hours Post-mortem
  • There are differences between donors as well, due to gender, age at death, certain medication, terminal coma, hypoxia, pyrexia, dehydration, stress or drugs and alcohol
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What have other studies shown between PMI and RNA?

A
  • Several other studies demonstrated that there is a clear correlation between RNA degradation and PMI
    o Under which circumstances those data have been obtained?
    o Is possible to come to consistent scientifically valued conclusion?
  • RNA remains largely intact for a considerable time depending on the tissue considered after death as long as bodies are stored at appropriate ambient conditions  measuring the level of RNA degradation can provide information about RNA quality in tissues, helping to establish a timeframe
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How can RNA be used to work out the cause of death?

A
  • Every cell in a tissue can show a different RNA transcript. RNA expression changes in response to external conditions. Therefore, traumatic events such as death leave a molecular mark in terms of shifter proportions of different transcripts within the mRNA pool
  • Ikematsu et al (2005) presented a first examples for the identification of possible biomarkers indicating certain causes of death: They examines gene expression changes in mice after two different causes of death, slow strangulation and fast execution using a guillotine
  • Four genes were identified that showed significantly different expression patterns between the two groups. Thus, these genes are candidate genes as possible biomarkers for strangulation
  • Recently, further candidate genes for the identification of methamphetamine related deaths and hypoxia related deaths were described. Additionally, works on contusion stress and mechanical asphyxiation were recently published
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

How can RNA be used to help predict the age of wounds?

A
  • One of the most important questions to be answered in forensic pathology is the age of external or internal wounds
  • Four main areas of interest:
    o In which order where the wounds inflicted?
    o Was the wound produced pre- or post- mortem
    o How long is the survival time after infliction of a lethal injury?
    o Is the investigated injury related to the incident?
  • Most of the recent investigations are based on changes within the wound margins
  • Promising results have been obtained for basic **fibroblast growth factor **(FGF family) which is peaking 48 hours after injury, e.g., in cerebral wounds, tissue-type plasminogen activator peaking at 1-hour post-injury and interleukin 10
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How can RNA be used to predict the age of stains?

A
  • Two studies have examined RNA degradation using special technique based on RT-PCR by comparing mRNA to rRNA in a single reaction
  • Currently, there is still no method available to assess the age of stains. In principle RNA fragmentation continues in vitro even if the material is completely dehydrated. However, this process is slow and shows considerable variation
  • The only possible differentiation so far is between recent (<1 year) and old (>8 years) stains
  • In the future, real time PCR or chip technology could offer a more sensitive approach although the main problem: the unpredictable conditions the stains are exposed
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How can RNA be used to predict the age of bloodstains?

A
  • Blood is the most found body fluid
  • Bauer at al 2003 investigated 106 bloodstains stored for up to 15 years
  • RNA distributed measured by lase-induced fluorescent capillary electrophoresis is closely correlated with the age of the samples
  • Bloodstains with age different of 5 years and more exhibit statistically significant variances in peak area of housekeeping genes in this study, b-actin and cyclophilin. RNA continues to be degraded in dried bloodstains, but it can be isolated from samples as old as 15 years
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What are the forensically relevant tissues in MRNA profiling?

Tell me about some presumptive tests for each

A

Saliva: Phadebas paper
Blood: Kasle-meyer
Sperm and seminal fluid: AP test (seminal fluid) the confirmatory microscopic testing for sperm
skin:

Have none: Urine, menstrual blood, vaginal secretions, sweat, skin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What are the pros and cons to presumptive testing?

A

* Pros
o Rapid
o Easy to perform
o Cost effective
o Quick results
o Could be done on the scene

*** Cons **
o Low specific, prone to false positive/ negative
o Interpretation is user dependant
o Non quantitative, only guidelines available
o Large area required and it could be destructive
o Cannot be automated
o Some body fluid still cannot be tested (e.g., menstrual blood, skin, sweat)
o Confirmatory tests not always available (saliva)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What can tissue-specific mRNA profiling help with?

A
  • Transcriptome analysis= is the analysis of the genes expressed in a cell
  • Every cell type will have a different pattern presented with different mRNA expressed according to the function to be developed by the specific tissue
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What are the stages to DNA/RNA co-extraction?

A
  • DNA/RNA co-extraction method
  • Swabs can be used for both methods (do not need a second collection)- means results are linked
  • DNA goes through normal profiling
  • RNA goes through different pathway
    o Remove DNase and RNase as they will start digesting RNA in extract
    o Reverse transcription- PCR doesn’t deal with no Thymine, therefore transcribe all in our solution and make into a DNA strand known as…
    o cDNA (complementary DNA) amplification (no uracil and now has thymine)
    o this cDNA can be amplified via normal methods
    o then both detected similarly
24
Q

How is RNA processed once extracted?

A
25
Q

What are some precautions to take for RNase degradation?

A
  • Maintaining RNase-free environment
  • Wear gloves! In LCN rooms it is also optimal to wear 2 pairs of gloves
  • Having an RNA-only working bench
  • Use RNase spray to inactivate RNase enzyme (DEPC)
  • Use your positive and negative as quality controls
  • Use internal positive controls
26
Q

Forensic samples are: Low quality, Low amount and may be influenced by external factors. What are some housekeeping genes?

A
  • Housekeeping genes are there no matter what the body fluid is and act as an internal positive control (need these due to the limitations of forensic samples as above)
  • Those involved in cycle of Krebs (needed in every cell)
27
Q

Tissue-specific mRNA markers

A
28
Q

What are some previously used mRNA markers, and why are they no longer used?

A
29
Q

Semen 5plex & singleplexes and Saliva multiplex

A
30
Q

Tell me about vaginal fluid and the markers for it?

A
  • Due to its epithelial nature, as saliva and skin could have similar gene expression patterns
  • Vaginal fluid uniquely have complex microenvironment and inter-individual variation shown maybe due to variation in menstrual cycle
  • Also, bacteria as lactobacilli could be microbial markers
    o HBD1
    o MYOZ1
    o CYP2B7P1
    o Ljen
31
Q

Tell me about menstrual blood and the markers for it

A
  • MB is one of the most challenging body fluid because of its similarity with blood, vaginal secretion and the high variation that occur during the cycle
  • During menstruation matrix metalloproteinase MMP are expressed in the endometrium
    o MMP7
    o MMP10
    o MMP11
32
Q

Tell me about skin and the markers for it

A
  • High attention has recently been given to skin because of the possibility to extract from traces touch DNA
    o LCE1C
    o CDSN
    o LOR
    o KRT9
33
Q

Tell me about the multiplex PCR

A
  • First multiplex mRNA system consisted of two markers per body fluid (e.g., blood, semen, saliva and vaginal secretion) (Jussola and Ballantyne, 2005)
  • Lidenbergh et al (2012) developed an end-point RT-PCR assay that simultaneously amplifies 19 mRNA markers specific for blood, semen, saliva, menstrual secretion, vaginal mucosa and skin and allows their differentiation
  • The multiplex also contains three general mucosa markers as well as three house keeping genes. Authors investigated not only the multiplex PCR assay performance and the specificity of the selected markers but also their sensitivity
  • Full RNA profiles were obtained with as little as 0.05µl of starting material whereas full DNA profiles were also observed when using at least 0.2µl
34
Q

Tell me about ‘TissueID 20plex’

A
35
Q

blood vs menstrual blood mRNA profile

A
36
Q

blood vs menstrual blood mRNA profile

A
37
Q

How is the electropherogram interpreted for mRNA profiling?

A
  • Netherland forensic institute (NFI) published their guidelines on how mRNA profiling was implemented in forensic casework, which we adopted in criminal casework in the UK. This procedure is also useful to avoid bias:
    o Bias; the researcher who performs the mRNA profiling should remain uninformed about the context of the case
    o mRNA results are generated separately following standard routing analysis
    o DNA results are generated separately following standard routine analysis
    o DNA/mRNA profiles are interpreted and a relationship between individuals and body fluids detected is established
    o Results are collated and conclusions are formulated in a final report
    o 4mRNA profiles are obtained for each sample
    o A standard mRNA results table was designed so that the results obtained form the mRNA replicated could be presented in categorised evaluations for all cell types represented in the 20 plex
    o X= being the number of signals of acceptable peak morphology and above detection threshold
    o N= is the number of times that a signal could have occurred
    o Issue when X is below the expected number /2 then use own judgement and see if it fits in the scenario being investigated
38
Q

RNA/DNA correlation

A
39
Q

Data interpretation table for mRNA profiling

A
40
Q

Look at practice case scenario

A
41
Q

Collaborative exercise mRNA-MGS part 1

A
42
Q

Collaborative exercise mRNA-NGS part 2

A
43
Q

What are the advantages of miRNA in forensics?

A

miRNA in forensics
* Hanson et al (2009) were the first to introduce miRNA profiling to forensic science. Firstly, they demonstrated that miRNA of subsequent quality and quantity can be extracted from forensic samples. Secondly, they investigated a total of 452 human miRNA molecules for use in body fluid identification

*** Advantages **
o Single stranded
o Small size
o More stable
o Less susceptive to degradation
o Greater discrimination power

44
Q

What were miRNA candidates used for?

A

miRNA candidates- could find the best markers for the body fluids

45
Q

Blood or semen sample?

A
46
Q

Tell me about DNA methylation and methylation patterns

A
47
Q

Targeted DNA methylation quantification

A
48
Q

Tell me about theSodium bisulphite conversion

A
49
Q

How are DNA methylation markers selected?

A
49
Q

How are DNA methylation markers selected?

A
50
Q

Whats the relationshio between DNA methylation and environment?

A
51
Q

How is DNA methylation used in forensics?

A
52
Q

Semen identification

A
53
Q

Semen identification

A
54
Q

How do you find tissue specific CpG sites?

A
  • Validation of reported suitable CpG sites in the literature
  • Analysis of the promotor region of tissue-specific genes
  • Analysis of genome-wide DNA methylation data
55
Q

Blood vs menstrual blood

A
56
Q

mRNA and DNA methylation profiling

A

7BBFM128: Forensic Biology (19 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions