C7 Flashcards
Sample preparation study design
- Problem
- Sampling
- Experimentation
- Hypothesis
- Data
- Report
Difference between sample preparation & sample pretreatment
Preparation
- Chemical modification
Pretreatment
- Physical modification
Impact of sample preparation/ pretreatment
- Time
- Cost
- Successful analysis
Explain specimen collection
- 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
Factor to consider in subject preparation, specimen collection & specimen handling
Subject preparation
- Drug regimen
- Physical activity
Specimen collection
- Sampling equipment
- Time of the day
Specimen handling
- Transportation
- Storage conditions
Sample preparation in molecular diagnostic
- Nucleic acid isolation
- Sample concentration
- Removal / inactivation of inhibitors
- Inactivation or removal of RNases
Importance of sample preparation
- Improved sensitivity
- Accurate identification of pathogen
- Improved reproducibility
- Reduce false positive
- Reduce false negative
Explain quality assurance
- 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
Controls in quality assurance
- Positive control
- Negative control
- Reagent blank
- Internal control
Sample considerations
- Age
- Type of tissue
- Homogeneity
Explain DNA extraction
- Carried out using lysis buffer
- Use CTAB, TPS & DNA extraction kit
2 parts of DNA protocol
- Technique to lyse cell gently & solubilise DNA
- Enzymatic/ chemical method to remove contamination protein , RNA or macromolecules
4 steps to remove & purify DNA
- Lysis
- Precipitation
- Wash
- Resuspension
Machines use for DNA extraction
- Mortar & paste: add liquid nitrogen
- Tissue lyser: large sample
Checking the quality of DNA
- 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
What is needed to check quality of DNA
- Buffer (TBE): provide ion to ensure electrical conductivity
- Agarose gel
- Power supply
- Gel chamber
Explain DNA quantification & its example
- Compare the concentration with Lamda HindIII DNA ladder
- Use Nanodrop Spectrophotometer for more accurate analysis
Explain the 260/280 & 260/230 ratio in nanodrop
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
Optimal 260/280 ratio for DNA & RNA
- DNA: 2.0
- RNA: 1.80
Explain total RNA purification
- Isolate RNA from other celullar component
- Inactive RNases
- Denature nucleic acid protein complex
- Cell or tissue disrupted
Explain RNases (Ribonucleases)
- Enzyme that degrade RNA
- Common lab contaminant
- Difficult to inactive
How to protect against RNase
- Wear gloves
- Use RNase free chemicals, tubes & pipette tips
- Add RNase inhibitors to reactions
Organic extraction of total RNA procedure
- 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
Affinity purification of total RNA
- 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
Absorbance of nucleic acid, protein & guanidine
- Nucleic acid: 260 nm
- Protein: 280 nm
- Guanidine: 230 nm
Formula of quantitation of RNA & sample concentration
RNA
- 1OD260 = 40 microlitre/ml of ssRNA
Sample concentration
- A260 x dilution x 40 = (RNA) microgram/ml
Estimating RNA purity by spectrophotometer
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
Evaluating RNA integrity
28S & 18S appear as distinct band with ratio 2:1
Function & characteristics of RNA gel
- Denature gel
- Denature loading dye
- RNase free
- Check for integrity of rRNA
What do we need to send
- Plant & animal genome sequencing
- RNA sequencing (Transcriptome sequencing)
- PCR product sequencing
Example of qualified DNA & RNA sample
- Information of DNA marker
- DNA sample with degradation
- DNA sample with RNA contamination
- RNA sample with protein contamination
Optimal 260/230 ratio
2 or above
