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Real-time PCR Real-time PCR

Real time and Quantitative PCR Stvarnom vremenu i kvantitativne PCR

Introduction to Real-Time PCR Introduction to Real-Time PCR

Real-time PCR is a type of quantitative PCR which measures the amount of cDNA or mRNA in a sample, either from a population of cells (tissue or cell culture), or recently even from a single cell. Real-time is used commonly to determine the expression of a gene's mRNA, and its expression levels (copy number of mRNA) during certain conditions (such as treating cells with a drug).  Real-time PCR can be used to compare normal (control) samples to disease samples, giving an idea as to expression changes which occur with pathogenesis.  Real-time PCR due to its sensitivity is also used in the detection of pathogens in the blood such as viruses. Real-time PCR je tip kvantitativna PCR koji mjere količinu mRNA u cDNA ili uzorak, ili od stanovništva stanica (tkiva ili kultura stanica), ili u posljednje vrijeme čak i iz jednog stanica. Real-time se obično koristi za odrediti izraz jednog gena u mRNA, i njegova izraza razinama (copy broj mRNA) za vrijeme određenim uvjetima (kao što su liječenja stanice s droge). Real-time PCR mogu se koristiti za usporedbu normal (kontrolu) na uzorcima bolesti uzoraka, davanje jedan ideji kao izraz za promjene koje se javljaju s patogeneze. Real-time PCR zbog osjetljivosti se također koristi u detekciji i patogeni u krvi kao što su virusi.

The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the Razvoj real-time PCR je kvantitativna varijabilnost eliminiran je tradicionalno povezana s kvantitativna PCR, na taj način omogućava se
routine and reliable quantitation of PCR products. rutinu i pouzdane quantitation of PCR products.

Table of Contents Table of Contents

Introduction to Real-time PCR Introduction to Real-time PCR

Real Time PCR: Advantages of Real-time PCR Real Time PCR: Prednosti Real-time PCR

History of Real-Time PCR History of Real-Time PCR

Real-time PCR systems Available Real-time PCR sustave naoružanja

Real-time PCR Protocols Real-time PCR Protocols

Mechanism of Real-time PCR Mehanizam Real-time PCR

Real Time PCR: Advantages of Real-time PCR Real Time PCR: Prednosti Real-time PCR

Each cell type expressess a set of mRNA molecules, known as a transcriptome.  In response to stimuli, cells are able to up-regulate or down-regulate factors such as protein enzymes inside the cell by regulating the mRNA levels of these factors.  mRNA levels are not always correlated with protein levels so some caution is necessary, however generally mRNA levels do loosely correspond to protein levels.  Measuring the mRNA levels of certain factors inside a cell using real-time pcr is a method which will give clues as to the amounts of these factors or proteins. Svaka stanica tip expressess skup mRNA molekule, poznate kao transcriptome. U odgovoru na osjeta, stanice su u mogućnosti da gore-dolje-regulirati ili regulirati čimbenike kao što je protein enzima unutar stanica koju uređuje mRNA razinama od tih faktora. MRNA razinama nisu uvijek korelirani s protein razinama tako neki oprez je potrebno, no općenito mRNA razinama učiniti loosely odgovaraju protein razinama. mjerenje razine mRNA određenih čimbenika unutar stanica pomoću real-time PCR je metoda koja će dati clues kao i na iznose od tih faktora ili proteina.

Traditionally, mRNA levels inside the cell were measured using Northern blotting.  This technique is regarded as a gold-standard in the measurement of gene expression/mRNA levels as it uses radiolabeled probe to label the RNA, which is then quantified using a scintillation counter giving very accurate readings. Tradicionalno, mRNA razinama unutar stanica su izmjeri pomoću Sjeverna upijajući. Ova tehnika se smatrati kao zlato-standard u measurement of gene expression / mRNA razinama kao što koristi radiolabeled sonda na oznaku, RNA, koja je zatim quantified koristeći scintilacijski brojač davanje vrlo točnih očitanja. Northern blotting although very accurate had several disadvantages including the use of radioactivity. Sjeverna upijajući iako je imao vrlo precizne nekoliko nedostataka, uključujući korištenje radioaktivnosti. Northern blotting required the accurate measurement of mRNA and total RNA from cells extracted in order to compare samples. Sjeverna upijajući potrebna je precizna mjerenja mRNA i ukupne RNA iz stanice ekstrakti, kako bi se usporediti uzorke. This was a problem because although detection methods were very accurate, error could be introduced into Northern blotting (or RNA dot/slot blotting) through differences in total RNA/mRNA levels between samples (ie cell treatments, different tissues, different animals, etc.).  It also required relatively large amounts of RNA which required large numbers of cells. To je bio problem, jer iako je otkrivanje metode su vrlo precizne, pogreška mogla biti uvedena u Sjevernoj upijajući (ili RNA dot / utor upijajući) kroz razlike u ukupnim RNA / mRNA razinama između uzoraka (tj. stanica tretmane, različita tkiva, različitih životinja, itd. ). Također je potrebno relativno velike količine RNA koji su potrebni veliki broj stanica. Recently, mRNA gene quantification or real-time pcr methods have been improved and are easier to perform and do not require the use of radioactivity.  Researchers often only have access to small amounts of cells especially in the fields of stem cell research and primary cell research, and real-time pcr has made it possible to quantify mRNA levels from even very small numbers of cells and lately even single cells.  However, this extreme sensitivity of real-time pcr methods makes it vital to protect one's samples from contamination, which would lead to false results. Nedavno, mRNA gena kvantifikaciji ili real-time PCR metode su poboljšani i lakše je izvesti i ne zahtijevaju korištenje radioaktivnosti. Istraživači često samo imati pristup male količine stanica posebno u poljima od kljun stanica istraživanje i primarna stanica istraživanje , I real-time PCR je to moguće kvantificirati razinu mRNA iz čak i vrlo mali broj stanica i stanica u posljednje vrijeme čak i jedinstveno. Međutim, ove ekstremne osjetljivosti real-time PCR metode važno je da se zaštitili jedan je od kontaminacije uzoraka, što bi dovesti do lažnih rezultata. Current real-time pcr methods and systems also do not require the measurement of mRNA or cDNA sample concentrations before real-time pcr is conducted. Trenutni real-time PCR metode i sustave i ne zahtijevaju mjerenje mRNA ili cDNA uzorak koncentracije prije real-time PCR je provedena.

History of Real-Time PCR History of Real-Time PCR

Higuchi et al.1,2 pioneered the analysis of PCR kinetics by constructing a system that detects PCR products as Higuchi et al.1, 2 pionir analizu PCR kinetika by izgradnja sustav koji detektira PCR proizvoda kao
they accumulate. oni nakupiti. This “real-time” system includes the intercalator ethidium bromide in each amplification reaction, Ovo "real-time" sustava uključuje intercalator ethidium bromid pojačanje u svakoj reakciji,
an adapted thermal cycler to irradiate the samples with ultraviolet light, and detection of the resulting fluorescence jedna prilagođena termalne cycler da irradiate uzorci s ultraljubičasto svjetlo, i otkrivanje ostvarenog fluorescencija
with a computer-controlled cooled CCD camera. s računalom upravljani hlađene CCD kameru. Amplification produces increasing amounts of double-stranded Pojačanje proizvodi sve veću dvostrukim nasukan
DNA, which binds ethidium bromide, resulting in an increase in fluorescence. DNA, koji povezuje ethidium bromid, što je rezultiralo u povećanju fluorescencija. By plotting the increase in fluorescence By plotting povećanje u fluorescenciji
versus cycle number, the system produces amplification plots that provide a more complete picture of the prema broju ciklusa, sustav proizvodi pojačanje zemljišta koje pružaju kompletnu sliku više od
PCR process than assaying product accumulation after a fixed number of cycles. PCR postupak od analiziranje proizvod akumulacije nakon fiksni broj ciklusa.

External sites: see Real Time PCR at Real Time PCR Info! Vanjske web stranice: vidjeti Real Time PCR u Real Time PCR Info!

References for Real-Time PCR Reference za Real-Time PCR

1. Higuchi, R., Dollinger, G., Walsh, PS, and Griffith, R. 1992. Higuchi, R., Dollinger, G., Walsh, PS, i Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Simultaneous pojačanje i otkrivanje specifičnih DNA sekvenci.
Biotechnology 10:413–417. Biotehnologija 10:413-417.

2. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Higuchi, R., Fockler, C., Dollinger, G., Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions. Kinetička PCR: realnom vremenu monitoring of DNA pojačanje reakcije.
Biotechnology 11:1026–1030. Biotehnologija 11:1026-1030.

Real-Time PCR Protocols Real-Time PCR Protocols	Real-Time PCR Bioinformatics and Databases Real-Time PCR bioinformatiku i baze podataka	Learn about Real-Time PCR Saznajte više o Real-Time PCR	Real-Time PCR Kits and Products Real-Time PCR Kits i proizvodi	Real-Time PCR Forum Real-Time PCR Forum	Real-Time PCR Books Real-Time PCR Knjige