1. BLOOD $ URINE COLLECTION
Set of blood collection tubes
1 x 4.5 ml. Citrate vacutainer
Citrated plasma
2 x 9 ml. EDTA vacutainer
DNA isolation and EDTA-plasma
1 x 2 ml. EDTA vacutainer
Measurement of HbA1c, hemoglobin and hematocrit
2 x 9 ml. Heparin vacutainer
RNA isolation
Urine collection set
2 x vacutainers, 1 x adaptor
Collection of urine
1 x 9 ml. Heparin vacutainer
Isolation of Leucocytes and heparin plasma

2. BLOOD & URINE COLLECTION
2 x 9 ml.
EDTA
vacutainer
2 x 9 ml.
Heparin
vacutainer
1 x 9 ml.
Heparin
vacutainer
1 x 4.5 ml.
Citrate
vacutainer
2 x 10 ml.
Urine
1 x 2 ml.
EDTA
vacutainer
Store in melting
ice during
transport
Store in melting
ice during
transport
Tube A:
Freeze within 1 hr.
Store in dry-ice
during transport
Tube B:
Add challenger
within 1 hr.
Store at 37°C during
transport
Store at room
temperature
during transport

3. EDTA: DNA & PLASMA BLOOD HANDLING 2 x 9 ml. EDTA vacutainer
Store in melting ice
during transport
Within 6 hr.
Centrifuge:
20 min G and 4°C
Vacutainer with
red cells, buffy-
coat and rest plasma
Collect plasma in
plastic tube
Vortex
shortly
<1 hr RT
Store at -20 °C
Division of plasma
18 Subsamples of ~500 µl
<1 hr RT
Snap-freezen
Store at <-30 °C
Methanol / Dry-ice

4. Heparine: RNA (rest & challenge)
BLOOD HANDLING
2 x 9 ml. Heparine vacutainer
TUBE A
TUBE B
<1 hr RT
<1 hr RT
Add challenger
3 subsamples of ~ 3 ml
Store at 37°C
during transport
Snap-freezen
After 6 hr. (exactly)
Methanol / Dry-ice
3 subsamples of ~ 3 ml
Store in dry-ice
during transport
Snap-freezen
Methanol / Dry-ice
After arrival at TNO
Store at<-60 °C
Store at<-60 °C

5. ** Year 2004 to Nov only

6. Array Based Genotyping
Costs must reduce further
The Sentrix Whole-Genome Genotyping BeadChip
100,000 SNPs
25,000 in transcripts
70,000 within 10kb of exons
Affymetrix 100K SNP Chips

7. AGRF Affymetrix Chip Genotyping Concordance and fail rates
Concordance comparison of samples with SNP fail rate of < 3%
Samples tested were an MZ twin pair, plus parents (S3 and S4).
Genomic, Buccal and MDA Genomic replicates were tested for each individual.
Sample MZ 2 Buccal was tested twice.
Fail - no call for one or both samples

8. Association Analysis Sharing between unrelated individuals
Disease alleles originate in common ancestor
High resolution
Recombination since common ancestor
Large number of independent tests
Powerful if assumptions are met
Same disease haplotype shared by many patients
Sensitive to population structure

9. Single Nucleotide Polymorphisms (SNP)
GGCTTCAGAATGGCC GGCTTCAAAATGGCC
Single base changes
Human SNPs = 9,856,125
- Validated SNPs 4,540,241
Frequency ~ 1 every 400 bp
Can cause functional changes

10. QIMR’s Sequenom MassARRAY Installation
(CCRC-E floor)

11. Multiplex Analysis
* A G
* A G
* T C
* A G
* C T

12. SNP Genotyping Minimum Finished Genotypes (>99%) Quality of DNA
Measure concentrations
Dispense in large volumes
Quality of Assays
Even peak heights
Test for Hardy-Weinberg equilibrium
Analysis of SNP data is particularly sensitive to assay problems
Genotype failures are not random
Heterozygous individuals fail most often
all SNP typing platforms
Error frequency of 0.11%
3268 DNA samples typed twice
159 pairs of MZ twins - No discordant genotypes

13. Twelve-Plex Genotyping

14. Whole Genome Association
Use DNA pooling to greatly reduce amount of genotyping
Possible now but reduced power
Use haplotypes to reduce number of SNPs that have to be genotyped
Haplotype blocks – HapMap project
Massive parallel genotyping
Costs must reduce further