Quantitative PCR (qPCR)

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The challenge


Nowadays, many researchers are interested on study of gene expression, DNA copy number measurement etc. There are some approaches to study gene expression level and quantify RNA. The most used method is Northern blot. In Northern blot, RNA is isolated and then separated by agarose gel electrophoresis and transferred to a membrane. Subsequently, it was probed with a specific DNA or RNA probe that is complementary to the gene of interest, and virtualized the gene expression level. Because RNA is non-amplified, Northern blot is low sensitivity and requires large amounts of RNA. It is the big disadvantage while studying gene, which have low expression level, or while sample limit (patient tumor sample, for example). Another method, which is more sensitive than Northern blot, combines Reverse Transcription and PCR (called RT-PCR). However, all of these techniques are difficult to obtain quantitative data, especially for low expressed genes.

The solution


In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. Now, there is a new technique developed to quantize gene expression, to determine initial number or low level of target DNA/RNA. This technique is called real-time PCR, also called quantitative PCR (qPCR). qPCR is based on the detection of the fluorescence signal produced by a reporter molecule, which increases as the reaction proceeds. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green) or sequence specific probes (i.e. TaqMan® Probes). With qPCR approach, one can study with little amount of nucleic acid and calculate relative gene expression accurately. qPCR can also be applied to detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence. With the technique developing, qPCR assays are now easy to perform, have high sensitivity, and more specificity.

The principle


In a qPCR, a fluorescent reporter is used to monitor the PCR as it progresses. The fluorescence emitted by the reporter as the PCR product accumulates with each cycle of amplification. Based on the molecule used for the detection, there are two type of qPCR technique:
 
  1. Non-specific detection: SYBR® Green is the most widely used double-strand DNA-specific dye reported for qPCR. Ethidium bromide can also be used for detection but its carcinogenic nature renders its use restrictive. Although these double-stranded DNA-binding dyes provide the simplest and cheapest option for qPCR, the drawback is that both specific and nonspecific products will emit signal.
  2. Specific detection target specific probes: Specific detection of qPCR use oligonucleotide probes labeled with both a reporter fluorescent dye and a quencher dye. Probes based on different chemistries are available for real time detection, these include: Molecular Beacons; TaqMan®; FRET Hybridization etc.
qPCR procedures

 
  1. RNA isolation from samples
  2. Reverse transcription
  3. Prepare and run qPCR.
  4. Data analysis: Primer design; qPCR procedures; excel file with Ct value, expression level (fold change) calculation
  5. Amplification plots and gene expression plots.

qPCR applications:

Quantitative mRNA expression studies.
 

DNA copy number measurements in genomic or viral DNAs
 

SNP genotyping.
 

Verification of microarray results.
 

ChIP (Chromatin IP) check and quantitative assay.
 

Drug therapy efficacy.
 

DNA damage measurement.
 


Please email our Project Manager, Ms. Areti Karadimos, at areti@biost.com with project requirements. A quote will be provided in less than 24 hours.