RT-PCR ANALYSISRT-PCR ANALYSIS

NOHA L. IBRAHIMNOHA L. IBRAHIM

WHY ReAL-TIMe PCR?WHY ReAL-TIMe PCR?

DISADvANTAge Of TRADITIONAL PCRPoor precision (Northern could even be

better) Low sensitivity Low resolution Non-automated Results are not expressed as numbers Ethidium bromide staining is not very

quantitative

WHY ReAL-TIMe PCR?WHY ReAL-TIMe PCR?

• ADvANTAgeS Of ReAL-TIMe PCRamplification can be monitored real-time wider dynamic range of up to 1010-fold no post-PCR processing of products (No gel-based analysis at the end of the PCR

reaction ⇒ high throughput)ultra-rapid cycling (30 minutes to 2 hours) highly sequence-specific

WHAT IS RT-PCR?WHAT IS RT-PCR?• Cells in all organisms regulate gene expression by turnover

of gene transcripts (messenger RNA, abbreviated to mRNA): The amount of an expressed gene in a cell can be measured by the number of copies of an mRNA transcript of that gene present in a sample.

• RT PCR reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously detect or quantify a targeted DNA molecule.

• During this reaction, fluoroprobes bind to specific target regions of amplicons to produce fluorescence during PCR. The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt filter flurometer.

WHAT ARe fLuOReSCeNT WHAT ARe fLuOReSCeNT DYeS?DYeS?• When a population of fluorochrome molecules is excited by

light of an appropriate wavelength, fluorescent light is emitted. The light intensity can be measured by flurometer or a pixel-by-pixel digital image of the sample.

• Excitation and Emission: Fluorodyes absorb light at one wavelength & thereby boosts an electron to a higher energy shell.

• The excited electron falls back to the ground state and the flurophore re- emits light but at longer wavelength.

• This shift makes it possible to separate excitation light from emission light with the use of optical filters.

• The wavelength (nm) where photon energy is most efficiently captured is defined as the Absorbancemax & the wavelength (nm) where light is most efficiently released is defined as the Emissionmax.

WHAT IS fLuOReSCeNCe WHAT IS fLuOReSCeNCe ReSONANCe eNeRgY ReSONANCe eNeRgY TRANSfeR (fReT)?TRANSfeR (fReT)?• FRET is a distance dependent interaction between the

excited states of 2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

• A donor chromophore, initially in its electronic excited state, then transfer energy to an acceptor chromophore. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance (1 / r6) between donor and acceptor, making FRET extremely sensitive to small changes in distance.

PCR STePSPCR STePS

1. Denaturation step: This step consists of heating the reaction to 90–95 °C. It causes DNA separation by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.

2. Annealing step: The reaction temperature is lowered to 50–65 °C allowing hybridization of the primers to the single-stranded DNA template.

3. Extension/Elongation step: At this step, the Taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction.

PROTOCOLS IN ReAL-TIMe PROTOCOLS IN ReAL-TIMe PCRPCR

• For analysis of gene expression levels, the RNA first needs to be transcribed into cDNA using a reverse transcriptase. Reverse transcriptases are enzymes generally derived from RNA-containing retroviruses. RT-PCR can take place in a two-step or one-step reaction.

• With two-step RT-PCR, the RNA is first reverse transcribed into cDNA using oligo-dT primers, random oligomers, or gene-specific primers. An aliquot of the reverse-transcription reaction is then subsequently added to the real-time PCR.

• Precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage

potocolss in real-time potocolss in real-time pcrpcr• In one-step RT-PCR — also referred to as one-tube RT-

PCR — both reverse transcription and amplification take place in the same tube, with reverse transcription preceding PCR. This is possible due to specialized reaction chemistries and cycling protocols. The fast procedure enables rapid processing of multiple samples and is easy to automate.

• The reduced number of handling steps results in high reproducibility from sample to sample and minimizes the risk of contamination since less manipulation is required. The advantages of each method are given below.

Two-step RT-PCR One-step RT-PCR ■ Multiple PCRs from one RT reaction ■ Easy handling ■ Flexibility with RT primer choice ■ Fast procedure ■ Enables long-term storage of cDNA ■ High reproducibility ■ Low contamination risk

sYper GreensYper Green

• When the SYBR Green binds to the double-stranded DNA of the PCR products, it will emit light upon excitation. The intensity of the fluorescence increases as the PCR products accumulate.

• This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding.

• However, since the dye does not discriminate the double-stranded DNA from the PCR products and those from the primer-dimers, overestimation of the target concentration is a common problem. Where accurate quantification is an absolute necessity, further assay for the validation of results must be performed.

• Nevertheless, amongst the real-time RT-PCR product detection methods, SYBR Green is the most economical and easiest to use.

taqman probestaqman probes• The two most popular alternatives to SYBR Green are

TaqMan and molecular beacons, both of which are relying on FRET for quantitation.

• TaqMan probes are oligonucleotides that have a fluorescent probe attached to the 5' end and a quencher to the 3' end.

• During PCR amplification, these probes will hybridize to the target sequences located in the amplicon and as polymerase replicates the template with TaqMan bound, it also cleaves the fluorescent probe due to polymerase 5'- nuclease activity.

• This separates the fluorescent and quenching dyes and FRET no longer occurs. Fluorescence increases in each cycle, proportional to the rate of probe cleavage.

molecular beacon molecular beacon probesprobes• Similar to the TaqMan probes, Molecular Beacons also

make use of fluorescent probes attached to the 5' end and a quencher attached to the 3' end of an oligonucleotide substrate, but FRET only occurs when the quenching dye is directly adjacent to the fluorescent dye.

• However, whereas the TaqMan fluorescent probes are cleaved during amplification, Molecular Beacon probes remain intact and rebind to a new target during each reaction cycle.

• When free in solution, the close proximity of the fluorescent probe and the quencher molecule prevents fluorescence.

• When Molecular Beacon probes hybridize to a target, the fluorescent dye and the quencher are separated resulting in the emittance of light upon excitation.

Scorpion probeSScorpion probeS

• The Scorpion probes, like Molecular Beacon, will not be fluorescent active in an unhybridized state, again, due to the fluorescent probe on the 5' end being quenched by the moiety on the 3' end of an oligonucleotide.

• With Scorpions, the probe and primer present as one molecule. However, the 3' end contain a primer, the 5' end contain a hairpin structure of probe and the loop is complementary to the target.

• A PCR blockers lies between the primer and the probe prevent the polymerase copying the probe.

• After amplification, heating cause the 3 primers to unfold, then cooling allowing the complementary probe to aneal which prevent the hairpin reformming again and then separate the fluorophore and quencher releasing fluorescence.

Multiplex probeSMultiplex probeS

• TaqMan probes, Molecular Beacons and Scorpions allow the concurrent measurement of PCR products in a single tube. This is possible because each of the different fluorescent dyes can be associated with a specific emission spectra.

• The term multiplex refers to the fact that detection of more than one pathogen is carried out simultaneously in one tube, by simultaneously amplifying multiple sequences in one single reaction.

• Not only does the use of multiplex probes save time and effort without compromising test utility, its application in wide areas of research such as gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and RNA detection, make it an invaluable technique for laboratories of many discipline.

baSic terMS uSed in rt-baSic terMS uSed in rt-pcrpcr• Baseline: The baseline is the noise level in early cycles,

typically measured between cycles 3 and 15, where there is no detectable increase in fluorescence due to amplification products.

• Background: This refers to nonspecific fluorescence in the reaction, for

• example, due to inefficient quenching of the fluorophore or the presence of large amounts of double-stranded DNA template when using SYBR Green I. The background component of the signal is mathematically removed by the software algorithm of the real-time cycler.

• Reporter signal: Fluorescent signal that is generated during real-time PCR by either SYBR Green I or by a fluorescently labeled sequencespecific probe.

baSic terMS uSed in rt-baSic terMS uSed in rt-pcrpcr• Normalized reporter signal (Rn): This is the emission

intensity of the reporter dye divided by the emission intensity of the passive reference dye measured in each cycle.

• Passive reference dye: On some real-time PCR instruments, the fluorescent dye ROX serves as an internal reference for normalization of the fluorescent signal.

• Threshold: The threshold is adjusted to a value above the background and significantly below the plateau of an amplification plot.

• Threshold cycle (CT) or crossing point: This is the cycle at which the amplification plot crosses the threshold, i.e., at which there is a significant detectable increase in fluorescence. The CT serves as a tool for calculation of the starting template amount in each sample

baSic terMS uSed in rt-baSic terMS uSed in rt-pcrpcr• Endogenous reference gene: This is a gene whose

expression level should not differ between samples, such as a housekeeping or maintenance gene.

• The use of an endogenous reference gene corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recovery, and differences in sample handling.

• Calibrator sample: This is a reference sample used in relative quantification.

• Standard curve: it is commonly generated using a dilution series of at least 5 different concentrations of the standard.

1a. Excitation filters 1b. Emission filters2. Tungsten halogen light source

Cycler

Real Time Detection

3. Microplate format(350 - 1000nm continuous)

Real Time PCR insTRumenTsReal Time PCR insTRumenTs

* Absolute quantificationIt uses serially diluted standards of known concentrations to generate a standard curve. The standard curve produces a linear relationship between Ct and initial amounts of total RNA or cDNA, allowing the determination of the concentration of unknowns based on their Ct values. This method assumes all standards and samples have approximately equal amplification efficiencies

* Relative quantification (relative fold change)Based on either an external standard or a reference sample, also known as a calibrator. When using a calibrator, the results are expressed as a target/reference ratio.

i. Relative standard method (using a standard curve and

then expressed relative to a single calibrator sample) ii. Comparative CT (2 -∆∆CT) method

3. TyPes of Real-Time PCR 3. TyPes of Real-Time PCR quanTifiCaTionquanTifiCaTion

3. PCR Quantification

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