Real Time PCR (qpcr) Dott. Finetti Luca

Transcription

1 Real Time PCR (qpcr) Dott. Finetti Luca

2 PCR (Polymerase Chain Reaction)

3 PCR (Polymerase Chain Reaction) Theoretically: 2 n

4 PCR (Polymerase Chain Reaction) It often used as a qualitative research approache Example: electrophoresis on agarose gel

5 Real Time PCR Amplification Quantification Concurrently! Gene expression GMO quantification in food

6 Expression studies: RT-PCR RNA extraction cdna synthesis

7 RNA extraction Phase separation Isopropanol precipitation Phenol (ph 4) Chloroform Aqueous phase Interphase Organic phase RNA pellet DNAse treatment!!!!!! Wash with ethanol 70 % Water elution

8 cdna synthesis Oligo-dT primer We are ready...?

9 What do we need besides cdna? Primers Tm: 60 C They are designed to amplify no more than 200 bp and, if possible, to extend an exon-exon junction.

10 How do we actually measure DNA? We use reagents that fluoresce in the presence of amplified DNA! 1) Ethidium Bromide Not used in real-time PCR because causes interferences in the amplification 2) SYBR Green I 3) Probes

11 SYBR Green I The limit is that SYBR Green is an unspecific dye 3 Taq Taq 3 l l l Taq 5 Detection of nonspecific amplifications Taq 3 l l When illuminated with light at 490nm, the SYBR+DNA complex fluoresces at 520nm.

12 Probe: TaqMan They can be designed in different colors or wavelengths and detect several different PCR targets in the same PCR tube.

13 To understand real-time PCR, let s imagine ourselves in a PCR reaction tube at cycle number 25 A soup of nucleotides, primers, template, amplicons, enzyme, etc. 1,000,000 copies of the amplicon (the amplified DNA product) right now.

14 What was it like last cycle, 24? Almost exactly the same, except there were only 500,000 copies of the amplicon. And the cycle before that, 23? Almost the same, but only 250,000 copies of the amplicon. If we were to graph the amount of DNA in our tube, from the start until right now, at cycle 25, the graph would look like this:

15 If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this: Plateau phase Linear phase Exponential phase

16 How can all this be used to measure DNA quantities?? Let s imagine that you start with four times as much DNA as I do picture our two tubes at cycle 25 and work backwards a few cycles. Cycle Me You ,000 1,000, ,000 2,000, ,000,000 4,000,000

17 So, if YOU started with FOUR times as much DNA template as I did Then you d reach 1,000,000 copies exactly TWO cycles earlier than I would!

18 What if YOU started with EIGHT times LESS DNA template than I did? Cycle Me You 25 1,000, , ,000, , ,000, , ,000,000 1,000,000 You d only have 125,000 copies right now at cycle 25 And you d reach 1,000,000 copies exactly THREE cycles later than I would!

19 So, if YOU started with EIGHT times less of DNA template as I did Then you d reach 1,000,000 copies exactly THREE cycles later than I would!

20 The Cq value represents the number of cycles needed to reach a set threshold fluorescence signal level. The exact level used for this threshold should be chosen so that it captures data during the exponential phase and is the same for all samples analyzed in a run

21 Actual Data Standard curve Reference genes ( or HouseKeeping)

22 Standard curve Relative quantification

23 Standard curve Cq y=a*x+b unknown sample Quality of cdna Primers and enzyme efficiency To calculate unknown samples Reliable data? Loq Q.

24 Relative quantification - ΔcT Ct Treatment Ctrl Tissue 1 (treated tissue) is 4 times more expressed

25 Why is it necessary to normalize the data? Difference in the extraction of mrna between samples Performance of reverse transcription PCR reaction

26 Normalization ΔΔcT (Livak et al. 2001)

27 Normalization ΔΔcT (Livak et al. 2001) Treatment Ctrl Ct Ct Tissue 1 (treated tissue) is 8 times more expressed

28 Normalization ΔΔcT and efficiency (Pfaffl et al. 2001)

29 Normalization ΔΔcT and efficiency (Pfaffl et al. 2001)

30 Normalization ΔΔcT and efficiency (Pfaffl et al. 2001) Treatment Ctrl Tissue 1 (treated tissue) is 7,5 times more expressed

31 Actual Data

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33 Thanks