Experimenttal part

Experiment

Absolute qPCR Relative qPCR

you will be provided with:1. sample A: plasmid of known concentration2. forward and reverse primers to detect the plasmid3. %E of the primers4. sample B:the same plasmid with unknown concentration

you will need to:find out the concentration of the sample B

you need to provide:1. RNA or cDNA from two samples you want to compare (sample 1 and sample 2)

2. sequences of primers for one gene of interest (GOI) and three reference genes

you will need to:1. estimate efficiency of your primers2. find out the amount of your GOI in sample 1

compared to sample 2

Standard 1

Standard 2

Standard 3

Standard 4

NTC

Sample B

Standard 5

Standard 6

Ref1 St 1

Ref1 St 2

Ref1 St 3

Ref1 St 4

Ref2 St 1

Ref2 St 2

Ref2 St 3

Ref2 St 4

Ref3 St 1

Ref3 St 2

Ref3 St 3

Ref3 St 4

GOI St 1

GOI St 2

GOI St 3

GOI St 4

Ref1 Sample 1

Ref2 Sample 1

Ref3 Sample 1

GOI Sample 1

Ref1 Sample 2

Ref2 Sample 2

Ref3 Sample 2

GOI Sample 2

For qPCR we will use DyNAmo Flash SYBR Green qPCR Kit (# F-415L, ThermoScientific.follow the link for Manual and other info)

For RT we will use Maxima First Strand cDNA Synthesis Kits for RT-qPCR (# K1642, ThermoScientific. follow the link for Manual and other info)

Kits and Protocols

Program

25 C 10'

60 C 40'

85 C 5'

4 C Hold

x1 reaction, V=20 ul Master mix for N reactions

MQ 9

5x buffer

4

Enzyme

2

RNA ? ul with 100- 1000 ng of total RNA

-

15 ul/reaction

Program

95C 7’

95C 10”40 cycles

60C 30” aquisition

95C

60C-95C 0.5C step aquisition for melt curve

x1 reaction, V=20 ul

Master mix for N reactions

x2 SYBR Green mix 10 ul

5 uM F primer 0.5 ul

5 uM R primer 0.5 ul

water 4 ul

template 5 ul -

15 ul/well

Absolute qPCR

sample A: 50 ul of plasmid, concentration 10 ng/ul

5 uM forward primer (F)5 uM reverse primer (R)

sample B: the same plasmid, concentration is unknownNTC= no template control= water

1. Use sample A to make 6 serial 10 fold dilutions.

Take into consideration:• each dilution will be checked in 3 identical

PCR reactions (technical replicates, a control for pipetting error)

• you will use 5 ul of template for each PCR reaction

2. Calculate the amount of qPCR reactions required and prepare corresponding volume of the Master mix (amount of

reactions + 2)

3. Pipet the Master mix into the plate (15 ul /well)

4. Add assigned templates to the Master mix in the wells

5. Seal the plate with the film. store at 4oC before run

6. Program the run

7. Perform the run

8. Your data file will be uploaded on the course web site

9. Analyse the data

10. Write a report

11. Make a presentation

1. Pool together x+y ul of our cDNA samples. If necessary dilute it up to 30 times (call this sample ”cDNA pool”).

Prepare a four serial dilutions of the cDNA pool

Take into consideration:• each dilution will be checked in 3 identical PCR

reactions (technical replicates, a control for pipetting error)

• you will use 5 ul of template for each PCR reaction

• try not to dilute your initial cDNA samples more than 300 times

2. Calculate the amount of qPCR reactions required and prepare corresponding volume of the Master mix (amount of reactions + 2)3. Pipet the Master mix into the plate (15 ul /well)4. Add assigned templates to the Master mix in the wells5. Seal the plate with the film. store at 4oC before run6. Program the run

7. Perform the run8. Your data file will be uploaded on the course web site

9. Analyse the data

10. Write a report

11. Make a presentation

Relative qPCR

sample 1: cDNA of your choicesample 2: another cDNA of your choice

5 uM Ref1 primers (forward and reverse)5 uM Ref2 primers (forward and reverse)5 uM Ref3 primers (forward and reverse)5 uM GOI primers (forward and reverse)