Quando si parla di PCR quantitativa si intende: A. Una PCR che produce grandi quantità di DNA B. Una PCR che emette quanti di luce C. Una PCR che quantifica il numero di molecole stampo presenti all inizio della reazione D. Una PCR che quantifica il numero di molecole stampo presenti alla fine della reazione X n = X 0 2 n PCR e PCR quantitativa X n = X 0 (1+E x ) n
Analisi prodotti PCR end-point Analisi prodotti PCR end-point X n = X 0 2 n Thermal cycler Real-time PCR Excitation filters X 0 = X n 2 n X 01 X 02 X 03 Optical module Sample plate Detector Emission filters X 0 = X nt 2 nt X nt threshold SYBR green is a dye that binds preferentially to double-stranded DNA changing its fluorescence properties. This effect allow to monitor the synthesis of DNA during real-time PCR reactions. nt 1 nt 2 nt 3
Cycle Threshold (Ct) Quantitative Real-time PCR The point at which the fluorescence rises appreciably above background has been called the threshold cycle! threshold Cycle
Absolute quantification Serial Dilutions of standard DNA template 10-2 10-3 10-4 10-5 10-6 Cycle Standard curve There is a linear relationship between the log of the starting amount of the template and its threshold cycle during real-time PCR, so giving known starting amounts of the target nucleic acid a standard curve can be constructed by plotting the log of starting amount versus the threshold cycle. Melt curve analysis Fluorescence Temperature 16S standard curve
df dt Melt curve There s only one peak, so a single PCR product! 10-2 10-3 10-4 10-5 10-6 Real Time PCR and Gene Expression analysis Real-time RT-PCR is a powerful tool in the study of gene expression Housekeeping Genes Real-Time RT-PCR The determination of gene expression levels requires the use of a gold standard - i.e. a gene whose expression level is not affected in the biological system being studied The HKG selected must have consistent expression in the experimental system being studied. The correct selection of the HKG for your quantitative real-time PCR reactions is essential to a successful assay. Due to the complexity of biological systems, there is not a single HKG that will serve the purpose of normalization for all assays. Real-Time RT PCR Housekeeping Genes are used to normalize experimental results for samples differences in the amount of total RNA added to each reaction for different efficiencies in reverse transcription beta actin,, GAPDH, beta2-microglobulin, ribosomal RNA, cyclophillin are some of the most used housekeeping gene in literature
Melt curve analysis Melt curve analysis Fluorescence Temperature df dt Gene of interest 16S 16S Gene of interest Gene expression Reference Sample a sample used as the basis for comparative results. The reference sample becames the 1x sample, and all other quantities are expressed as n-fold difference relative to the reference sample.
Sample X Ct = C T GOI -C T HKG GOI : gene of interest C T GOI Sample X Ct 2 - Ct = C T GOI -C T HKG HKG : housekeeping gene C T HKG Sample Ref = 2 - Ct = C T GOI -C T HKG X 0 = X nt 2 n Example: C T GOI = 28 C T HKG = 25 X 0GOI X 0HKG = 2 CT HKG = 2 - Ct = C T GOI -C T HKG 2 CT GOI Ct = C T GOI -C T HKG = 3 X 0 GOI 1 = 2-3 = X 8 0HKG Example: Ct X = 2 1 Ct Ref = 3 Ct X = 4 2 Ct Ref = 3 Relative expression = 2 - C T Ct = -1 Relative expression = 2 CT = 2 1 = 2 Ct = 1 Relative expression = 2 CT = 2-1 = 1/2 = 0.5 Gene expression Relative quantification If the efficiencies are 100%, or at least they are high and comparable: Relative expression = 2 - C T Relative quantification Ct Sample Type GOI Ct HKG Ct Ct S Ct-R Ct Relative expression 1 (Reference) 28 25 3 0 1 2 25 23 2-1 2 3 24 26-2 -5 32 4 27 24 3 0 1 Ct = GOI Ct-HKG Ct Ct = S Ct - R Ct=Sample Ct-Reference Ct Relative Expression= 2-(S Ct-R Ct)
Total RNA extraction Reverse transcription with random primers Quantitative realtime PCR amplification with specific csgb oligos Quantitative realtime PCR amplification with specific flic oligos Quantitative realtime PCR amplification with specific 16S oligos csgb or flic Gene Expression 16S Relative quantification Ct Sample Type GOI Ct HKG Ct Ct S Ct-R Ct Relative expression 1 (Reference) 28 25 3 0 1 2 25 23 2-1 2 3 24 26-2 -5 32 4 27 24 3 0 1 Ct = GOI Ct-HKG Ct Ct = S Ct - R Ct=Sample Ct-Reference Ct Relative Expression= 2-(S Ct-R Ct)