Seminar 3

Pipetting qPCR

Primer design

1. Primers must be specific to your GOI
2. All primers used on the same plate should have similar Tm
3. Design primers to anneal efficiently at 60°C (Tm should be about 65°C).

Tm = the temperature at which one half of the DNA duplex will dissociate to become single stranded

http://insilico.ehu.es/tm.php?formula=basic

Should I design my primer to the coding sequence?

No, design them to a sequence present in your template and not present anywhere else

If oligodT were used for cDNA synthesis it might be wise to design primers closer to the 3’ end of the template

Why give preference to the 3’ end?��Reverse transcription is not perfect

TTTTTTT

Reverse transcription is not perfect

TTTTTTT

cDNA

Ideally all mRNA should be copied from end to start

Reverse transcription is not perfect

TTTTTTT

cDNA

In real life Reverse transcriptase might get tired and distracted

Reverse transcription is not perfect

TTTTTTT

cDNA

In real life Reverse transcriptase might bump into a secondary structure (hairpin) on your mRNA

Reverse transcription is not perfect

cDNA

Primers annealing at the 3’ will have a greater chance to catch all copies: complete and incomplete

• Has a mix of oligo(dT) 18 + random hexamer primers => high sensitivity in low copy number transcript detection assays. Random priming is incapable of distinguishing between mRNA and other RNA species present in the reaction (rRNA, tRNA, non-coding RNA)

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• Synthesis not at 37 or 40 C, but at elevated temperatures (50-65°C) => destabilizing secondary structures on mRNA

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• It is desirable to add equal amount of cDNA for all samples during qPCR

Group 3 (Ezgi and Aimer)� cDNA preparation

Use the total RNA mount as a proxy for equilibrating cDNA amount between samples

RIN = RNA Integrity Number >5 => RNA is intact

How to assess cDNA concentration?

Primer-blast vs Primer 3 vs manual

Primer3Plus

Has elaborate explanations for all values that you see in the Primer-blast

Splicing variants for EF1 alpha mRNA from Rainbow Trout

Primer-Blast has limitations depending on the origin of the template

• The corresponding RefSeq genome or just genome database was not available in Primer –Blast=> no way to check primer specificity

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• Rena has NGS data for the same samples -> use custom primer alignment on your own NGS data

Considering Azotobacter genomic sequence

• Primer –Blast was complaining about exon/intron information being not available
• => design primers for junction sites manually

Rainbow trout genes have annotated exons and introns

Protocols

2X DyNAmo Flash SYBR Green qPCR Kit master mix:

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• hot-start version of a modified Tbr DNA polymerase
• SYBR® Green I
• PCR buffer
• 5 mM MgCl2
• ROX is optional
• dNTP mix including dUTP�

From Thermus brockianus

Pipette at RT

Activate for 7’ at 95C

ROX passive reference dye

qPCR machines with lamp-based excitation (excitation/emission in all wells simultaniously)

ROX passive reference dye

qPCR machines with LED-based excitation (excitation light source moves from well to well/emission is detected in individual wells)

2X DyNAmo Flash SYBR Green qPCR Kit master mix:

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• hot-start version of a modified Tbr DNA polymerase
• SYBR® Green I
• PCR buffer
• 5 mM MgCl2
• ROX is optional
• dNTP mix including dUTP

Preventing contamination with previous PCRs�by using dUTP in the nucleotide mix�

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• If you run qPCRs regularly, amplicons might spread around and contaminate water, primers, plastic, bench => get mixed into your qPCR reactions and skew the results

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Solution => kill the amplicons:

• At the first PCR dUTP will be incorporated into the amplicon
• Uracil-DNA glycosylase (UDG) can digests dU-containing DNA
• Mix your qPCR normally except adding UDG and the pre-run step to your qPCR program (50 C for 2’) => digest sneaky amplicons that might have gotten into your qPCR reactions

Pushing the limits!�Reaction volume 15 ul

• 20 - 50 μL is recommended for most real-time instruments.

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• The minimum possible V depends on the real-time instrument and pipetting skills.

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• The reaction volume can be increased if a high template amount is used

How would this help?

REACTION SETUP AND CYCLING PROTOCOLS

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• Perform the reaction setup in an area separate from nucleic acid preparation and PCR product analysis

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• As the hot-start DNA polymerase is inactive during PCR setup, it is not necessary to do the setup on ice

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• Pipette with sterile filter tips

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• Minimize the exposure of the qPCR master mix to light (photobleaching)

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• Minimize pipetting errors by using calibrated pipettes and by preparing premixes to avoid pipetting very small volumes

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• Use optically clear caps or sealers to achieve maximum signal

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• Use a cap sealing tool or firm finger pressure to close caps properly, or use a film sealer

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• Avoid touching the optical surface of the cap or sealing film without gloves, as fingerprints may interfere with fluorescence measurements

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• Use powder-free gloves

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• Centrifuged plates before starting the program to force the solution to the bottom of the tubes and to remove any bubbles

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• Use molecular biology grade H2O

15 ul reaction

7.5 ul

None

3 ul (<200ng)

Depends on your stock

Your primers will arrive with suggestion to dissolve them till 100 pmol/ul concentration

Your primers will arrive with suggestion to dissolve them till 100 pmol/ul concentration

Recommended final concentration for the primers 0.5 uM

Your primer stock will 100 pM/ul

= 100 uM

=> Dilute your primer stock 200 x to achieve the final concentration of 0.5 uM

If genomic DNA is used as a template, use a 30 s annealing/extension time.

What is the benefit of detecting the signal at extension T = 72 C?

What starting temp for Melt curve makes sense?

Hot-start pol will not amplify anything without this step!

Hot-start pol will not amplify anything without this step!

Why to have the final extension step?

Group 1 qPCR

Duncan, Natnael, Qingxuan

Plate

GOI 1: ATG8f, GOI 2: ATG8h, GOI 3: ATG8i, REF 1: UBQ, REF 2: PP2A

Protocol

Melt curve spans 22.5 C (0.3C increment) => 75 steps, each 40 sec long = 3000 seconds = 1 hour?

Default in the CFX suggest to use 0.5C step for 5 sec each: 22.5 C(0.5 C increment) = 225 sec = 4 minutes

Final extension step?

= 41 cycles

Required volume and composition for mastermixes

• 1000 ng of total RNA used for cDNA synthesis in 20uL in reverse transcription reaction mix
• Reverse transcript reaction to be further diluted 30x.

Primer concentration and amount in ul?

NB!! Template DNA is not included into the MM!

Will you use the same MM for all 96 wells in your plate?

Group 2

Julia and Rena

Dilution factors for the Standards

Technical replicates annotation

6 NTC controls?

Final extension step?

Master mix

1. For each primer set -> calculate how many reactions will you need to pipette?

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• Create a Master mix for the corresponding number of reactions + 4 (loss to plastic surfaces)

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• Each of your master mixes should contain SYBR green mix + Fw primer + RE primer+ MQ

Group 3

Ezgi and Aimer

55C for annealing, what about extension? NB! polymerase is active at 60-72C

No Melt curve?

Without the 1^st step: 7’ at 95C the hot start polymerase will remain inactive

Group 4

Ashwath and Mohammad

I have not found any files for protocol, plate layout or master mix calculation

Group 5

Casey and Philip

No Melt curve?

Principle of quantification by qPCR

How much of specific DNA sequence do I have in the sample?

original concentration

Absolute: copy number or ng

Relative: fold difference between samples

(primers /probes ensure specificity towards your sequence of interest)

qPCR question

y = a*x +b

For qPCR dilutions we often use 10x dilutions

• Easy to plot x axis as log₁₀

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X = log starting quantity

Y= Ct value

b = y-int

a = slope