qpcr course Alyona Minina Department of Plant Biology SLU
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1 qpcr course Alyona Minina Department of Plant Biology SLU 2014
2 What do you want to know? Data Analysis and interpretation Lecture 2 Statistical analysis of qpcr How qpcr works Lecture 2 Application of qpcr Lecture 2 Basics Lecture 1 Normalization Troubleshooting More Lecture 2 Everything
3 Results of the quiz 1. Did you learn anything new from the lecture A lot yes no If yes, do you think you will remember it tomorrow? No some (not the equations) yes, after revising it yes Would you prefer to have more information in the lecture, keep it the same, reduce it? Reduce/split in 2 parts Keep the same More information a requests for less calculations abbreviation of each symbol 4. Were the tasks too easy, ok, or too hard? too hard ok too easy will be simplified 9 6 0
4 Results of the quiz 6. was/would be the homework helpful Nope a bit Yes Would you suggest any changes? it was a bit too fast/i need more time to think more group work Application part was too fast and not clear
5 I. Efficiency II. Quantification Absolute qpcr Relative qpcr III. Primer design IV. The practical part
6 Why a short product? the biggest issue seem to be the efficiency ( see the 72 o C for 5 minutes step in end point PCR) than shorter is the amplicon than less its amplification is susceptible to suboptimal conditions depending on the kit, there might be a risk of running out of components during the reaction time issue. you want your results asap
7 primer annealing denaturation each cycle extension fluorescence detection Please draw one cycle and write the temperatures for each one Helicase-dependent isothermal DNA amplification. Vincent et al.,embo Reports. 2014
8 Can you PCR RNA? What is the difference between end point and real-time PCR? What is absolute qpcr? What is relative qpcr? Why do you need to normalise your PCR results? What are the ways to normalise them?
9 CtAc=20 A gene control sample CtRc=25 A gene treated sample Reference gene control sample what does it tell you? Reference gene treated sample fluorescence what are the units for this? what is this? why does this look so bad? what s this? what are the units for this? what are these? CtAc CtAt CtRc CtRt cycles
10 how to make it look linear? fluorescence why? is it linear? why? why? cycles
11
12 what is log? log a b=c => a c =b log 3 9=? log 2 8=? log 749 =? log 5 1=? log 10 16=? => =16 log 10 8=? => =8 log 10 4=? log 10 2=? => =4 => =2
13 What is primer Efficiency? What does it mean when Efficiency is 100%? What does it mean when E=2? How can it be more than 100%? Why can it be less than 100%? How to estimate efficiency?
14 DNA E = 100% Ct=20 is it important to know concentration of the initial sample? dilute twice DNA Ct=21 dilute twice DNA Ct=22 dilute twice DNA Ct=23
15 C t =23 X Δx = relative concentration in log scale Δy = ΔC t Δy/Δx= slope C t =22 X E = 10 1/slope % Efficiency = (E 1) x 100% C t =21 X C t =20 X axis x is relative concentration in log scale where do these numbers come from? why in log scale again?
16 DNA E =? E =? E =? E =? Ct=20 Ct=19 Ct=20 Ct=33 dilute five times DNA Ct=22 Ct=22 Ct=23 Ct=35 Ct=24 Ct=25 Ct=24 Ct=36.8 Ct=26 Ct=27 Ct=25 Ct=39 group 1 group 2 group 3 dilute five times DNA dilute five times DNA group 4
17 DNA E =? E =? E =? E =? Ct=20 Ct=19 Ct=20 Ct=33 dilute five times DNA Ct=22 Ct=22 Ct=23 Ct=35 Ct=24 Ct=25 Ct=24 Ct=36.8 Ct=26 Ct=27 Ct=25 Ct=39 group 1 group 2 group 3 dilute five times DNA dilute five times DNA 3. If you have time calculate E using the data of other groups as well step 1? set 4. If you have time calculate also R2 group 4 why it is not important to know the true concentration, again? amount of DNA in the undiluted sample to your favourite number step 2? determine the log10 of your DNA concentration step 3? calculate the slope=δy/δx or? linear function y = kx + m Manually: slope
18 Linear regression calculation ou can also find this video on the website of the course
19 DNA E =? E =? E =? E =? Ct=20 Ct=19 Ct=20 Ct=33 dilute five times DNA Ct=22 Ct=22 Ct=23 Ct=35 Ct=24 Ct=25 Ct=24 Ct=36.8 Ct=26 Ct=27 Ct=25 Ct=39 group 1 group 2 group 3 Calculation of Efficiency dilute five times DNA dilute five times DNA 3. If you have time calculate E using the data of other groups as well step 1? set 4. If you have time calculate also R2 group 4 why it is not important to know the true concentration, again? amount of DNA in the undiluted sample to your favourite number step 2? determine the log10 of your DNA concentration step 3? calculate the slope=δy/δx or? linear function y = kx + m Manually: slope step 4? calculate E = 10-1/slope step 5? if you want % calculate (E-1)*100% step 6? CHECK if your result MAKES SENSE how would you do it?
20 group 1 group 2 group 3 group 4 amount of DNA log10 of the DNA amoount x y x y x y x y ini-al DNA sample 625 2, , , , , st 5 fold dilu-on 125 2, , , , , d 5 fold dilu-on 25 1, , , , , ,8 3d 5 fold dilu-on 5 0, , , , , slope k - 2,8614-3,8628-2,2891-2,8327 R^2 1 0, , ,99857 E=10^(-1/slope) E 2, , , , %E=(E-1)*100% %E 123, , , , " 20" 10" 0" y"="$2.8614x"+"28" R²"="1" 0" 1" 2" 3" 30" 20" 10" 0" y"="$3.8628x"+"30" R²"=" " 0" 1" 2" 3" 30" 20" 10" y"="$2.2891x"+"27" R²"=" " 0" 0" 1" 2" 3" 40" 38" 36" 34" y"="$2.8327x"+"40.9" R²"=" " 32" 0" 1" 2" 3"
21 Efficiency You can also find this link on the website of the course
22 Absolute qpcr what is absolute qpcr? how do you calculate the amount of DNA in your sample? what is the difference between the standard curve for absolute qpcr and for primer Efficiency? can you use the same steps for calculating?
23 Calculation of Efficiency vs Absolute qpcr standard curve is it applicable for absolute qpcr standard curve? step 1 set amount of DNA in the undiluted sample to your favourite number step 2 determine the log 10 of your DNA concentration step 3 calculate the slope= y/ x or? linear function y = kx + m slope
24 axis y is Ct???? C t =23 X y = kx + m x =? C t =22 X C t =21 X C t =20 X axis x is quantity in log scale
25 Relative qpcr what is relative qpcr? how do you make sure that your samples have the same amount of total DNA?
26 what is delta? Ct method For PCR exactly the same amount of DNA was used from sample A and from sample B The gene of interest (GOI) was detected. How much more/less of this gene is present in the sample A sample A sample B what is the possible disadvantage of this approach? how would you try to solve it? normalized to what? CtA CtB Normalized expression ratio = E Ct = E (CtA -CtB)
27 Ct why do you think there are two deltas here? sample A sample B 1. Normalize expression of gene of interest in A and B E GOI - C t GOI = E GOI -(C t A-C t B) what will it tell you? C t A C t Ar C t Br C t B 2. Normalize expression of reference gene in A and B E ref - C t ref = E ref -(C t Ar-C t Br) what will it tell you? 3. Calculate expression ratio of GOI and reference gene E genea - C t GOI /E ref - C t ref =?
28 Primer design do you have suggestion, what to take into consideration?
29 DyNAmo Flash SYBR Green qpcr Kit optimal amplicon size more than 50 bp, but less than 250 bp GC content as close to 50% length: not shorter than 18 ntp, preferably not longer than bp is usually good. both primers should have the same melting T (Tm) check for self annealing and primer dimer formation check for specificity, allow several mismatches you can eliminate or detect genomic DNA contamination make sure, that all your primers are written down as 5 ->3 (including the reverse one) T/A on 3 end might decrease not specific amplification try Primer3 and BLAST-primer softwares (on the course site)
30 reference genes you want them to be equally expressed in control and test samples: check available literature and ask people around check available microarray data (GENEVESTIGATOR, the link to it is on the website of the course, you will need to create an account to use it) figure out what genes are involved in key pathways in all cells present in your sample avoid picking genes within the same pathway or cross-talking pathways pick only single member family genes choose the least conservative coding part of a gene you picked it is on your conscience to trust single reference gene data. just for you information, good people use more than one reference.
31 On the website of the course you can download the Excel file for primer order Please fill it in and send to me not later than 4 pm on the 7th of October (next Tuesday)
32 The practical part
33 Experiment Absolute qpcr Relative RT-qPCR my sample and my primers your samples and your primers
34 Experiment layout Absolute qpcr Relative RT-qPCR Standard 1 Ref1 St 1 Ref3 St 1 Ref1 Sample 1 Standard 2 Ref1 St 2 Ref3 St 2 Ref2 Sample 1 Standard 3 Ref1 St 3 Ref3 St 3 Ref3 Sample 1 Standard 4 Ref1 St 4 Ref3 St 4 GOI Sample 1 Standard 5 Ref2 St 1 GOI St 1 Ref1 Sample 2 Standard 6 Ref2 St 2 GOI St 2 Ref2 Sample 2 Sample B Ref2 St 3 GOI St 3 Ref3 Sample 2 NTC Ref2 St 4 GOI St 4 GOI Sample 2
35 Absolute qpcr You will get from me: 1. Sample A = an eppendorf with a plasmid of known concentration 2. Sample B = an eppendorf with the same plasmid with concentration known only to me 3. primers to detect the plasmid and data about their E You will need to: here 1. Run an absolute qpcr home 2.find out the concentration of the plasmid in the sample B home 3. write a report in a form of Results + Materials and Methods + Figure with Legend for an article in a given journal home 4. make a short presentation of your results + stuff you want to share
36 Relative RT-qPCR You will need to: home home here here here home home home home home home 1. Have RNA from 2 samples you want to compare 2. Design 4 pairs of primers to detect 1 gene of your interest and 3 reference genes in your samples 3. Run RT 4.Run primer efficiency test for all 4 primer pairs 5. Run PCR on your samples using all 4 primer pairs 6. Calculate primer efficiency 7. Assess performance of your reference genes. Pick 2 best. 8. Use these 2 reference genes to normalize your data using C t and C t methods 9. Make up data to perform a statistical analysis 10. Write a report in a form of Results + Materials and Methods +Figure with Legend for an article in a given journal 11. include results into your short presentation
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