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MIC661:MOLECULAR DIAGNOSTIC > C7 > Flashcards

C7 Flashcards

1
Q

Sample preparation study design

A
  • Problem
  • Sampling
  • Experimentation
  • Hypothesis
  • Data
  • Report
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2
Q

Difference between sample preparation & sample pretreatment

A

Preparation
- Chemical modification

Pretreatment
- Physical modification

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3
Q

Impact of sample preparation/ pretreatment

A
  • Time
  • Cost
  • Successful analysis
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4
Q

Explain specimen collection

A
  • Special collection system design for collection of microbes
  • DNA & RNA will be damage in lysed
  • Need to avoid contamination that can cause false positives
  • Important to quantify & use equipment & reagents
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5
Q

Factor to consider in subject preparation, specimen collection & specimen handling

A

Subject preparation
- Drug regimen
- Physical activity

Specimen collection
- Sampling equipment
- Time of the day

Specimen handling
- Transportation
- Storage conditions

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6
Q

Sample preparation in molecular diagnostic

A
  • Nucleic acid isolation
  • Sample concentration
  • Removal / inactivation of inhibitors
  • Inactivation or removal of RNases
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7
Q

Importance of sample preparation

A
  • Improved sensitivity
  • Accurate identification of pathogen
  • Improved reproducibility
  • Reduce false positive
  • Reduce false negative
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8
Q

Explain quality assurance

A
  • Specimen integrity is crucial to avoid incorrect result
  • Ensure the absence of product in amplification techniques is due to absence of target rather than presence of inhibitor
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9
Q

Controls in quality assurance

A
  • Positive control
  • Negative control
  • Reagent blank
  • Internal control
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10
Q

Sample considerations

A
  • Age
  • Type of tissue
  • Homogeneity
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11
Q

Explain DNA extraction

A
  • Carried out using lysis buffer
  • Use CTAB, TPS & DNA extraction kit
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12
Q

2 parts of DNA protocol

A
  • Technique to lyse cell gently & solubilise DNA
  • Enzymatic/ chemical method to remove contamination protein , RNA or macromolecules
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13
Q

4 steps to remove & purify DNA

A
  • Lysis
  • Precipitation
  • Wash
  • Resuspension
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14
Q

Machines use for DNA extraction

A
  • Mortar & paste: add liquid nitrogen
  • Tissue lyser: large sample
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15
Q

Checking the quality of DNA

A
  • Poor DNA will not perform well in PCR
  • Mix 10microlitre of DNA with 10 microlitre loading buffer
  • Load the mixture into 1% agarose gel
  • Analyse the result
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16
Q

What is needed to check quality of DNA

A
  • Buffer (TBE): provide ion to ensure electrical conductivity
  • Agarose gel
  • Power supply
  • Gel chamber
17
Q

Explain DNA quantification & its example

A
  • Compare the concentration with Lamda HindIII DNA ladder
  • Use Nanodrop Spectrophotometer for more accurate analysis
18
Q

Explain the 260/280 & 260/230 ratio in nanodrop

A

260/280
- Indicate how pure the sample from contaminating protein
- Protein absorb at 280nm, low 260/280 indicate presence of high amount of protein

260/230
- How pure sample from salts & other contaminats
- Eg: EDTA, Phenol

19
Q

Optimal 260/280 ratio for DNA & RNA

A
  • DNA: 2.0
  • RNA: 1.80
20
Q

Explain total RNA purification

A
  • Isolate RNA from other celullar component
  • Inactive RNases
  • Denature nucleic acid protein complex
  • Cell or tissue disrupted
21
Q

Explain RNases (Ribonucleases)

A
  • Enzyme that degrade RNA
  • Common lab contaminant
  • Difficult to inactive
22
Q

How to protect against RNase

A
  • Wear gloves
  • Use RNase free chemicals, tubes & pipette tips
  • Add RNase inhibitors to reactions
23
Q

Organic extraction of total RNA procedure

A
  • Lyse cells
  • Add phenol:chloroform:isoamyl alcohol to lyse sample & centrifuge
  • Organic phase (bottom) separates from aqueous phase (top- total RNA)
  • Remove RNA solution to clean tube, precipitate RNA & wash with ethanol
24
Q

Affinity purification of total RNA

A
  • Lyse cell & spin to remove large cell debris
  • Apply lysate to column with glass membrane
  • Wash with alcohol to remove contaminants
  • Treat with DNase to remove contaminating DNA
  • Apply water to column to purify RNA washes off the glass
  • Collect the RNA
25
Q

Absorbance of nucleic acid, protein & guanidine

A
  • Nucleic acid: 260 nm
  • Protein: 280 nm
  • Guanidine: 230 nm
26
Q

Formula of quantitation of RNA & sample concentration

A

RNA
- 1OD260 = 40 microlitre/ml of ssRNA

Sample concentration
- A260 x dilution x 40 = (RNA) microgram/ml

27
Q

Estimating RNA purity by spectrophotometer

A

A260/A280
- Pure RNA with range of 1.8-2.0
- 1.7 indicate protein contamination

A260/A230
- Properly purified RNA range of 1.8-2.0
- 1.7 indicate guanidine contamination

28
Q

Evaluating RNA integrity

A

28S & 18S appear as distinct band with ratio 2:1

29
Q

Function & characteristics of RNA gel

A
  • Denature gel
  • Denature loading dye
  • RNase free
  • Check for integrity of rRNA
30
Q

What do we need to send

A
  • Plant & animal genome sequencing
  • RNA sequencing (Transcriptome sequencing)
  • PCR product sequencing
31
Q

Example of qualified DNA & RNA sample

A
  • Information of DNA marker
  • DNA sample with degradation
  • DNA sample with RNA contamination
  • RNA sample with protein contamination
32
Q

Optimal 260/230 ratio

A

2 or above

MIC661:MOLECULAR DIAGNOSTIC (7 decks)

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