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qPCR measures quantities of target sequences
Targets can be DNA, cDNA or RNA
DNA: present in a sample ?
number of copies in a sample ? -> copy number analysis
! Plasmids need to be linearized for qPCR
cDNA: quantify expression levels -> gene expression analysis
expression = amount of mRNA in a sample
mRNA
cDNA to prevent degradation
reverse transcription
RNA: quantify expression levels of small non-coding RNA -> miRNA profiling
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0009545
qPCR protocol involves many steps
Growing the samples
RNA extraction
DNase treatment
cDNA synthesis
Primer design
qPCR
Starting with good samples: RNA extraction
mRNA or miRNA extraction ?
-> different kits
recommended: use a kit that can extract both e.g. Qiagen miRNeasy
use same samples for mRNA and miRNA profiling
Check RNA quality
-
Integrity: RNA breaks down easily
-
Purity: RNA can contain inhibitors of qPCR reaction
! Don’t aim for high quality, aim for equal quality between samples !
Checking RNA integrity by microfluidic electrophoresis
e.g. Agilent Bioanalyzer, Bio-Rad Experion
Evaluates integrity of 18S and 28S rRNA
You look at rRNA while you’re interested in mRNA
If you see these peaks there’s no degradation
Checking RNA integrity by 5’-3’ mRNA ratio
Perform reverse transcription on RNA
Use qPCR to measure HPRT1 in cDNA using two primer sets
HPRT1
5’
Cq 3’
Cq 5’
No degradation: Cq 5’ = Cq 3’
Degradation:
Cq 5’ <<< Cq 3’
HPRT1 is a reference gene with stable, low expression levels
HPRT1 is expressed in all organisms
AAAAAAA
3’
Inhibitors distort the qPCR reaction
No inhibitors
Inhibitors: shift Cq to right
e.g. phenol, ethanol...
Checking RNA purity by SPUD assay
SPUD = potato gene with no homology to any known sequence
Add equal amounts SPUD to each RNA sample
Create controls:
positive: SPUD + heparin (a known inhibitor)
negative: SPUD + water
Perform qPCR to measure SPUD
SPUD
+
water
SPUD
+
heparin
SPUD
+
RNA1
SPUD
+
RNA2
SPUD
+
RNA3
Cq=22
Cq=27
Cq=22
Cq=26
Cq=22
Clean samples have same Cq as water
Samples with inhibitors have higher Cq
ΔCq > 1: presence of inhibitors
Starting with good samples: DNase treatment
To remove genomic DNA contamination from mRNA samples
Recommended but never 100% efficient
Kits: Qiagen RNase-Free DNase Set for DNase digestion during RNA purification
Solutions:
• Intron or exon-exon junction spanning primers: not always feasible
gDNA will generate a longer or no PCR product
• No RT control to detect gDNA contamination
Starting with good samples: cDNA synthesis
To transform RNA into more stable cDNA
Prepare all cDNAs that you are going to use in a single batch
Kits: Biogazelle uses BioRad iScript Advanced
Primers: Biogazelle uses a mix of random and oligodT primers
Validation:
RNA
RNA
1/4
RNA
1/16
RNA
1/64
RNA
1/256
cDNA
cDNA
cDNA
cDNA
cDNA
Cq=18
Cq=20
Cq=22
Cq=24
Cq=26
Create RNA dilution series
Synthesize cDNA
qPCR
Cqs follow dilution series: good cDNA synthesis
cDNA synthesis + DNase treatment directly on cells: Ambion Cells-to-CT kit
Different ways to measure amount of PCR product
Fluorescent dyes:
e.g. Sybr Green, EvaGreen...
Fluorescent dye binds to ds DNA
Denaturation: dye is released and fluorescence reduces
Polymerization: primers anneal and ds PCR product is formed
Dye binds to ds product and fluorescence increases
Fluorophore-containing DNA probes:
e.g. Taqman...
Plot of the fluorescent signal (Rn) during PCR
Rn
Signal of unbound dye
Cycle
Threshold for the transition of baseline to exponential phase
∆Rn = Rn -baseline
ABI instruments use different thresholds for different plates
! Not Ok: Use the same threshold on every plate !
qPCR generates Relative Quantities (RQ)
more DNA/RNA
less DNA/RNA
∆Cq = difference between 2 Cq’s
0
1
2
3
4
10 11...
Detection of abnormal amplification: visual inspection of the curves
Inspection of melting curves
Inspection of melting curves
qPCR primer design guidelines regarding PCR products
degraded material: 50-80 bp
good material: 80-150 bp
avoid repeats and domains (high conservation: no specific primers)
take into account splice variants and SNPs
qPCR primer design software
Primer Design
Primer3Plus: http://primer3plus.com/cgi-bin/dev/primer3plus.cgi
PrimerQuest: https://eu.idtdna.com/PrimerQuest/Home/Index
Primer-BLAST: http://blast.ncbi.nlm.nih.gov/
Checking primer specificity
BLAST: http://blast.ncbi.nlm.nih.gov/
BiSearch: http://bisearch.enzim.hu/
Checking secondary structures in the PCR product
UNAfold: http://mfold.rna.albany.edu/
Checking location of SNPs
UCSC in silico PCR + SNPs track: http://genome.ucsc.edu/cgi-bin/hgPcr
UNAfold secondary structure prediction of PCR product
Secondary structure overlapping primer annealing site
Do not use these primers !
qPCR primer design guidelines regarding primers
intron or exon-exon junction spanning
length: 9-30 bp (ideally: 20 bp)
melting temperature Tm: 58-60 °C (ideally: 59°C)
maximum Tm difference between primers: 2°C
GC content: 30-80% (ideally: 50%)
5 nucleotides at 3’ end < 3 G or C
avoid runs > 3 identical nucleotides
Same primers ordered from different vendors will give different results
-> variation in amount of inhibitors in the primers
Recommended: IDT primers
Checking primer / probe specificity
Primer specificity
Always: inspection of melting curves
First time use: gel analysis
Probe specificity
Negative control (sample in which gene is not expressed)
Take home message: overview of recommendations
Different location pre- and post PCR
96 well versus 384 well
cheaper
more data
easier to pipet smaller volumes
Manual versus robotic pipetting
96 well
384 well
multichannel pipets: calibrate regularly
! Never use volumes < 1 µl (pipetting errors too high)
Check quality of pipets: pilot experiment with same sample-target in all wells
-> differences in Cq should be < 0.2
Take home message: overview of recommendations
Replicates
Choose biological replicates over technical replicates
4 biological replicates
Controls
Negative controls: no template, no RT, biological control
Positive control: biological control
Reference genes
Minimum 3
Validate primers, kits and reference genes before the experiment !
Take home message: overview of recommendations
Sample maximization
Put all samples of the same gene on the same plate
No need to repeat reference genes on each plate
When you want to compare one gene over different conditions
<-> when you want to compare genes: gene maximization
If samples of same gene are to be spread over different plates: use IRCs