One Cycle Ahead – when Q
stands for quick in qPCR with its
design and running
outperforming expectations
Agilent Technologies qPCR eSeminar
Robert Loewe
GeneWake GmbH
July 30, 2015
For Research Use Only. Not for Use in Diagnostic Procedures
1
What’s on the agenda
Minor Notes on the side:
Gene Expression:
Things to ponder in the darkest hour
Not all experiments are created equal
Bisulfite Treated DNA:
A cautionary tale
HRM:
From Design to Outcome
Multiplex:
The more the merrier?
Impact of Instrumentation:
How data can look like and how to interpret
For Research Use Only. Not for Use in Diagnostic Procedures
Minor Notes on the side
For Research Use Only. Not for Use in Diagnostic Procedures
3
Difficult samples for qPCR
Minute amount of starting material (down to single cells)
Degraded NA (FFPE, bisulfite converted DNA)
Presence of Inhibitors in isolated NA (NA from Urine)
For Research Use Only. Not for Use in Diagnostic Procedures
4
First know your qPCR assay
Efficiency
 efficient PCR comes earlier
Product (size, melting point …)
 smaller amplicon can have
faster PCR
Limit of detection
 amount near limit of detection
might need more cycles and Cq’s
between replicates vary
For Research Use Only. Not for Use in Diagnostic Procedures
5
Quantitative PCR – dilution series
For Research Use Only. Not for Use in Diagnostic Procedures
Quantitative PCR – Limit of detection
For Research Use Only. Not for Use in Diagnostic Procedures
Secondly adjust your qPCR settings
Minute amount of starting material (down to single cells)
 carrier NA
Degraded NA (FFPE, bisulfite converted DNA)
 short amplicons, short initial heating
Presence of Inhibitors in isolated NA (NA from Urine)
 dilute NA, design new primer, clean up
change isolation method
For Research Use Only. Not for Use in Diagnostic Procedures
8
Gene Expression
For Research Use Only. Not for Use in Diagnostic Procedures
9
Prostate cancer set-up
Biopsy
Gleason Scoring
biopsy – FFPE
clinical data analysis
RNA isolation
gene expression profiling
Surgery
tissue from surgery – FFPE
blood (Paxgene) and urine
from day of surgery
RNA isolation
gene expression profiling
Molecular Profiling
Gene expression - Gleason Score
patient stratification
For Research Use Only. Not for Use in Diagnostic Procedures
Bioinformatics
For Research Use Only. Not for Use in Diagnostic Procedures
11
Biopsy analysis (FFPE) with gene expression profiling
For Research Use Only. Not for Use in Diagnostic Procedures
Blood analysis with gene expression profiling I
BPH
For Research Use Only. Not for Use in Diagnostic Procedures
PCA
13
Blood analysis with gene expression profiling II
For Research Use Only. Not for Use in Diagnostic Procedures
14
Bisulfite treated DNA
For Research Use Only. Not for Use in Diagnostic Procedures
15
Bisulfite conversion – as a difficult example
For Research Use Only. Not for Use in Diagnostic Procedures
16
Inhibition Effect I
1:2 dilution bisulfite treated DNA
For Research Use Only. Not for Use in Diagnostic Procedures
17
Inhibition Effect II
1:4 dilution bisulfite treated DNA
For Research Use Only. Not for Use in Diagnostic Procedures
18
Inhibition Effect III
1:10 dilution bisulfite treated DNA
For Research Use Only. Not for Use in Diagnostic Procedures
19
Inhibition Effect IV
Undiluted bisulfite treated DNA – different kit
For Research Use Only. Not for Use in Diagnostic Procedures
20
Inhibition Effect V
Undiluted bisulfite treated DNA – different kit
For Research Use Only. Not for Use in Diagnostic Procedures
21
HRM
For Research Use Only. Not for Use in Diagnostic Procedures
22
Introduction
High Resolution Melting is viable method to screen for SNPs, InDels and Sequence Changes
In essence it is working with intercalating dyes that insert themselves into the double strand
Slow melting while making high resolution measurements results in a melting curve
Temperature is gradually increased to find melting point of the amplicon
Mismatches in the Sequence (e.g. Heterozygotes) show up as a “dent” in the melting curve
PCR product remains intact and can be recycled
Usable for high throughput screening
For Research Use Only. Not for Use in Diagnostic Procedures
What to detect
SNPs:
e.g. two Alleles A and B
(pretending that in human genetics only
homozygotes and heterozygotes exist)
possible formats to measure
Somatic Mutations:
100%
A
50%
A & 50% B
100%
B
(Homozygote for A)
(Heterozygote)
(Homozygote for B)
Everything from 0 – 100% is theoretically possible
For Research Use Only. Not for Use in Diagnostic Procedures
EvaGreen
 Known Facts
 Things to remember
For Research Use Only. Not for Use in Diagnostic Procedures
EvaGreen: Things to remember
EvaGreen can induce primer dimers
More does not equal better!
Perfect amount in extremely trial and error
For Research Use Only. Not for Use in Diagnostic Procedures
26
Primer-Design
 Know your sequence!!!! Be aware of SNPs flanking your region of interest (roi)
 Try to limit yourself in regards to size
60-300bp is working for Homozygote/Heterozygote distinction in general, in difficult
sequences shorter amplicons are better also for somatic mutations but too short gives lower
fluorescence level
 Be aware of primer dimers and multimers
 Design more than one set if possible
 Maybe use melting probe for (e.g. small oligo that specifically binds to your roi)
 Be Aware of Pseudogenes
For Research Use Only. Not for Use in Diagnostic Procedures
Example: SNP rs4988235 (lactose intolerance)
AGCTGGGTATTCACAAGGATGTATGTGAAAAAACTCACCATGCCATACATTTCCCTTTTTATAAATTATACCTCAGTC
ACACTGTAAAAAAAAGCCAAACCCCCTTTTCAAAGACGACCTTACATCAAACCTATTAATAAAACTAGGAAAAAG
CAGGGCTGCTTTGGTTGAAGCGAAGATGGGACGCTTGAATGCCCTTTCGTACTACTCCCCTTTTACCTCGTTAATA
CCCACTGACCTATCCTCGTGGAATGCAGGGCTCAAAGAACAATCTAAAAATCAAACATTATACAAATGCAACCTAA
GGAGGAGAGTTCCTTTGAGGCCAGGG
[G/A]
CTACATTATCTTATCTGTATTGCCAGCGCAGAGGCCTACTAGTACATTGTAGGGTCTAAGTACATTTTTCCTGAATGA
AAGGTATTAAATGGTAACTTACGTCTTTATGCACTCTATAAACTATGACGTGATCGTCTCCGTCTAACAACTACACTC
AAATGCTTACCAAGCTCTTTAAAGGGAAGAATTCCATGGTCGTATGAGCATTCAACAGTTACATAAAAATGTATTT
GCAGTGAATTCTAGTATGTCCCATACCAAAGATTAAAAACATGCAACAAATCTGTTTATCTCTGCTCTCATCATATCA
AAGTGACTCTGATACCTCATCACAGCAGTGCATCCGAGCCATAGCTTC
18 Forward primers and 10 reverse primers
60 – 653 bp amplicon length
For Research Use Only. Not for Use in Diagnostic Procedures
28
Chemistry
 Run protocol is essential
 Input amount of samples to be compared should be equal
 Primer amount should be sufficient but not too high
 Subsequent results will be generated with the AriaMX or the
LightCycler480 using the Agilent HRM Ultra-Fast Loci Master
For Research Use Only. Not for Use in Diagnostic Procedures
For Research Use Only. Not for Use in Diagnostic Procedures
Initial Denaturation 3 minutes
Cycling: e.g.
10s 95°C
15s 60°C
25s 72°C
Or
10s 95°C
30s 60°C
In Fast Mode
5s
95°C
10s 60°C
30
Some Pictures
 Different sizes




17/1
12/2
5/2
2/5
69 bp
112 bp
187 bp
278 bp
 Different Percentages
 0%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, 100%
For Research Use Only. Not for Use in Diagnostic Procedures
69 bp
112 bp
5% Detection
32
Picture Perfect
Initial Denaturation
3 minutes
Cycling:
10s at 95°C & 30s at 60°C
Multiplex
For Research Use Only. Not for Use in Diagnostic Procedures
34
Multiplexing is not always the solution, but is sometimes
the answer to too much work in too little time.
35
Impact of Instrumentation
For Research Use Only. Not for Use in Diagnostic Procedures
36
Important facts needed
•
•
•
•
•
Homogenous plate cycling is essential
Inter / Intra plate variation
Plate calibrator needed for variable Cq interrun
Standardized Cq calling advisable
Unbiased scanning of different wells is
important
• Rapid and precise cycling vital
For Research Use Only. Not for Use in Diagnostic Procedures
37
Thank
you
for
spending your time
with me and special
thanks
to
Agilent
Technologies for letting
me talk
[email protected]
WWW.GENEWAKE.COM
For Research Use Only. Not for Use in Diagnostic Procedures