Q-PCR QUANTITATIVE-PCR 세포생물학및실험 2 박태식교수님

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1 Q-PCR QUANTITATIVE-PCR 세포생물학및실험 2 박태식교수님

2 Title : Study that the Drug A inhibits the accumulation of fat. Schedules 1. Mouse necropsy : take up the blood and major tissues(organs) ex) heart, liver, fat, muscle 2. Check the gene expression about fatty acid oxidation under the RNA level(rna prep, cdna synthesis, Q-PCR) 3. Check under the protein level

3 Quantitative-PCR quantitative PCR(qPCR), also known as a real-time polymerase chain reaction (RT-PCR), is a laboratory technique of molecular biology based on the polymerase chain reaction(pcr). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (quantitative real-time PCR).

4 Northern blotting for RNA - Northern blotting is often used to estimate the expression level of a gene by visualizing the abundance of its mrna transcript in a sample. - Disadvantages 1) It requires relatively large amounts of RNA and provides only qualitative information of mrna levels. 2) Estimation errors arising from variations in the quantification method can be the result of DNA integrity, enzyme efficiency and many other factors. For this reason a number of standardization systems (often called normalization methods) have been developed. Some have been developed for quantifying total gene expression, but the most are for the specific gene being studied in relation to another gene called a normalizing gene.

5 Normalizing gene is selected for its almost constant level of expression. These genes are often selected from housekeeping genes as their functions related to basic cellular survival normally imply constitutive gene expression. This enables researchers to report a ratio for the expression of the genes of interest divided by the expression of the selected normalizer, thereby allowing comparison of the former without actually knowing its absolute level of expression. The most commonly used normalizing genes are those that code for the following molecules : tubulin, albumin, cyclophilin, and ribosomal RNAs, β-actin.

6 Quantitative PCR Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA ex) SYBR green (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. ex) TaqMan

7 Non-specific detection vs. Specific detection Non-specific detection Specific detection Reporter double-stranded DNA-binding dyes fluorescent reporter probes Method - A DNA-binding dye binds to all dsdna in PCR, causing fluorescence of the dye. - An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle - The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. - The close proximity of the reporter to the quencher prevents detection of its fluorescence - breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. - An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.

8 Non-specific detection vs. Specific detection Disadvantag e Advantage Non-specific detection - bind to all dsdna PCR products, including nonspecific PCR products (such as Primer dimer) ; potentially interfere with, or prevent, accurate monitoring of the intended target sequence. - only one target sequence can be monitored in a tube - only needing a pair of primers to carry out the amplification, which keeps costs down. Example SYBR green TaqMan Specific detection - More expensive than non-specific detection method - Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube. - prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR

9 SYBR green vs. TaqMan SYBR Green TaqMan specificity < expense <

10 Imagining Real-Time PCR(Ct value) - The value that represents the cycle number where the amplification curve crosses an arbitrary threshold.(ct : Threshold cycle number) - Ct values are directly related to the starting quantity of DNA, by way of the formula : amplification of quantity DNA = 2^Ct - The smaller/faster Ct is, the larger is amplification of quantity DNA (inverse proportion). - Ct : Ct target gene Ct reference gene - Ct : Ct Sample - Ct Control

11 Protocol(Q-PCR) 1. complementary DNA(cDNA) ; 1/10 dilution Ex) DDW 180 ul cdna 20 ul 5 ng/ul Total 200 ul 2. Add 4ul of each 5ng/ul cdna in the 96 well PCR plate(20ng of cdna in a tube) β-actin CPT 1A High Fat Diet 1조 2조 3조 4조 5조 6조 1조 2조 3조 4조 5조 6조 Fenofibrate Compound A_5mg Compound A_10mg Compound B_5mg Compound B_10mg One group - 6 conditions (HFD / Feno / A_5mg / A_10mg / B_5mg / B_10mg) - Primer about two kinds of gene (β-actin; housekeeping gene, CPT1A; target gene)

12 Protocol(Q-PCR) 3. Make the pre-mixture. Components volume Primer Forward (10pmol/ul) 0.2 ul Primer Reverse (10pmol/ul) 0.2 ul 2X SYBR green 10 ul DDW 5.6 ul 5 ng/ul cdna 4 ul Total 20 ul/reaction Components Primer Forward Primer Reverse 2X SYBR green DDW N + 2 = ul 1.6 ul 80 ul 44.8 ul Make two kinds(β-actin, CPT1A) of pre-mixture. (i.e. using two E-tube) 4. Put 16 ul of pre-mixture into the 96well plate and pipetting. (CAUTION! DO NOT MIX between a sample and other sample.) 5. Cover with film for Q-PCR on the 96 well PCR plate.

13 Protocol(Q-PCR) 6. Centrifuge 1000 rpm for 2 min. 7. Operate the Q-PCR Denaturation Annealing Extension Cycle Total time Condition 95, 15sec 60, 15 sec 72, 45 sec 40 cycle About 2.5 hour

14 Results (Draw a table and vertical bar graph ) 1. Write the Ct value in the table 2. Set the range 3. Draw graph(vertical bar graph) 1 3-1) 3-2) 3-3) 2

15 Results form (Draw a table and vertical bar graph ) <Table> 1) my group (1 replicate) 2) total group (6 replicate) Ex) <Graph> 1) Group 1 2) Group 2 3) Group 3 4) Group 4 5) Group 5 6) Group 6 7) Total (6 replicate)

16 Discussion 1. Explain that the effects of compound A and B about the accumulation of fat in liver under the mrna expression. So, write your opinion about the possibility of compound A/B as drug for fatty liver disease. 2. Calculate the amplification of quantity Gene X in group A and B. What is difference between group A and B on the relative expression level of gene X? Ct value Target gene (Gene X) A (Control) B (Sample) Reference Gene (β-actin)