week 2: sample QC Flashcards
SAMPLE QC WORKFLOW
SAMPLE RECEIVED
project managers assign QC task
SAMPLE
RETRIEVE
SAMPLES TO BE
PROCESSED PLACE
ON SAMPLE QC
SHEET
for DNA:
DNA SAMPLE
DILUTED
(10X)
Qubit
Analysis
Gel
Electrophoresis
Data
Analysis
for RNA
RNA SAMPLE
ALIQUOT &
DENATURE
2100 Bioanalyzer
(Plant & Animal Assays)
DNA
CONTAMINATION
(YES OR NO)
if yes - Gel
Electrophoresis then DNA analysis then OMS report
if no - Data
Analysis then OMS REPORT
Overview
Gel electrophoresis - method for separation and analysis of DNA, RNA, and Proteins and their
fragments, based on their size and charge.
Bioanalyzer – An Agilent instrument that is a chip-based capillary electrophoresis machine to
analyse RNA, DNA, and protein. Used for RNA QC
Nanodrop – A Thermo instrument (spectrophorometer) used to quantify concentrations. Ok to
check contamination levels and 260/280 ratios. Can’t distinguish DNA from RNA
Qubit – A flourometer used to quantify concentrations
260/280 or 260/230 – the purity of your sample
Agilent 2100 Bioanalyzer
1 - load sample
- ready to use reagent kits
- quick start instructions
- chip prep. in less than 5 minutes
- sample volumes in the ul range
2 - run analysis
- start analysis at the press of a button
- minimal use of hazardous chemicals and waste disposal
3 - see data
- predefined protocols
- automated data analysis
HOW DOES IT WORK?
Pin-electrodes create
electro-kinetic forces,
driving fluids or
molecules within fluids
through micro-channels
to perform
electrophoretic
separations.
How to interpret RNA results?
what is RIN
use the Sample QC Standard for RNA Examination:
sample grade either pass or fail
pass:
- RIN > or equal to 4.0
- total sample amount: > or equal to 200 ng
- sample concentration: > or equal to 20 ng/ul
- sample purity: no sample degradation, no genomic contamination
fail:
- RIN < 4.0
- total sample amount: < 200 ng
- sample concentration: < 20 ng/ul
- sample purity: 5s rRNA region
degradation, Low 28S peak, Possible genomic DNA contamination, Severe sample
degradation, Degraded Genomic DNA
The RNA Integrity Number (RIN) is a numerical value that measures the quality and integrity of RNA samples
What to Look for ?
- Amount(ug)
- RIN Value
- Electropherograms
HOW TO CALCULATE AMOUNT(UG)
Concentration(ng/ul) x Volume(ul)/1000 = Amount (ug)
- Concentration(ng/ul) = Bioanalyzer results
- Volume(ul) = Volume check
- 1000 = Conversion Factor
Bioanalyzer introduction
The Bioanalyzer software automatically
generates the ratio of the 18S to 28S ribosomal subunits
RNA degradation
Result Notes/Call: Degradation observed
Description: Decay of isolated RNA leads to fragmentation of 18s and 28s
Conclusion: FAIL if ratio of area of degradation is high relative to the area of 18s and 28s
peaks
Low 28s Peak
Result Notes: Low 28s Peak
Description: Occurs when the 28s Peak is less than 50% of the 18s Peak.
Conclusion: Please determine the corresponding conclusion IF the initial conclusion
5S Peak Degradation
Result notes: 5S Peak Degradation
Description: Occurs when the 5S peak is more than 50% of the 18S Peak
Conclusion: Please determine the corresponding conclusion
DNA contamination
Result Notes/Call: DNA contamination detected
Description: Usually occurs when there is a lift after the 28S Peak
Conclusion: FAIL if genomic DNA is detected
How to Interpret DNA results?
use Sample QC Standard for DNA Examination
sample grade either pass or fail
sample grade: pass
- gel image: Clear gDNA bands and no
Degradation or Contamination, Smear phenotype for MDA products from single cell and genomes
- total sample amount: > or equal to 200ng
- sample concentration: > or equal to 10ng/ul
- sample purity: No gDNA degradation, No protein/RNA contamination
sample grade: fail
- gel image: Visible gDNA Bands and
Degradation and contamination, No Visible gDNA Bands and Degradation and contamination
- total sample amount: < 200ng
- sample concentration: < 10ng/ul
- sample purity: Minor gDNA
Degradation, Moderate gDNA
Degradation, Minor protein
contamination, Severe gDNA
Degradation, Moderate protein
contamination
