REAL TIME PCR

Quantitative PCR is a variant of the polymerase chain reaction (PCR) used to simultaneously amplify and quantify the amplification product (DNA segments, commonly called the amplicon). The measurement is per cycle, so it is called real-time PCR. The quantitative PCR (qPCR) is possible because the amplicon is marked with a fluorophore that, after being excited at a certain wavelength, is detected by the sensors of the thermocycler and this allows the quantification of the emitted fluorescence.

Service Information

Quantification strategies:

Absolute Quantification: It determines the exact number of DNA molecules (qPCR) or RNA (qRT-PCR) present in a given sample.
Relative Quantification: It is not necessary to know the precise amount of the sequence of interest.

Service Specifications

Generation of cDNA.
Test of oligonucleotides.
Generation of the standard curve per gene (to be evaluated).
Real Time PCR Reactions.
Analysis of differential expression.

Sample Requirements

At least 30-40 μg of total RNA is required for each of the conditions to be evaluated.
RNA samples should be delivered lyophilized or in water.
The client must provide information of the quotient D.O. 260/280 and D.O. 230/260, obtained at the time of the quantification of the samples. In both cases must be ≥ 2.0.

Recommendations for RNA purification and extraction

Users are advised that RNA extraction is carried out with phenol and that the extracted RNA is subjected to a subsequent purification step using silica gel columns.

The Genomic Services laboratory also offers a RNA extraction service.

Quantification

It is suggested that the quantification be performed by spectrometry or fluorometry, with the idea of achieving a measurement as accurate as possible. The client must provide information of the quotient D.O. 260/280 and D.O. 230/260, obtained at the time of the quantification of the samples, in both cases must be ≥ 2.0.

Requirements for OligoNucleotides (primers)

The recommended degree of purity for them is HPLC, however, in most cases, only desalted oligonucleotides can be employed. The service request format must be submitted along with the samples and oligonucleotides.

Sending Samples

Samples must be submitted under the above conditions. Samples can be stored in any kind of tube, properly labeled (matching the name in the format), and perfectly sealed.

Delivery of Results

The results generated will be sent to the client via email in an Excel file, the data delivered include:

The images corresponding to the dissociation curve and the Tm for each of the amplicons.
The established dynamic ranges guarantee the efficiency of the PCR reaction.
In differential expression analysis, relative to the selected treatment as calibrator.