Biomedical Engineering Reference
In-Depth Information
Reverse transcription PCR (RT-PCR)
PCR is used to detect DNA sequences but it can be adapted to identify
RNA in a sample as well. To this end, reverse transcriptase is added
together with a reverse primer and nucleotides to generate a cDNA. The
sample, or an aliquot of the sample, is then subjected to standard PCR
procedures. This has proved to be a relatively simple and rapid method
to determine if a particular gene is expressed in a cell or cell population.
Semiquantitative PCR
PCR is extremely sensitive and can easily amplify a single DNA seg-
ment. However, standard PCR is not quantitative. While in theory there
should be a relationship between the amount of starting material and
the final product, in practice this is not always the case. Semiquantitative
PCR was established to overcome this deficiency. The development of
Semiquantitative and quantitative PCR has been an especially signifi-
cant advancement in our ability to ascertain the level of expression of a
gene in different cells and tissues.
A common method of generating semiquantitative RT-PCR data is to
analyze the products after varying numbers of PCR cycles (Figure 5).
This allows the investigator to identify the quantitative portion of the
amplification cycles. In the example shown, the products are visualized
every 3 cycles of PCR beginning at 21 cycles, so that the amount of DNA
generated should increase 8-fold between samples. By comparing the
amount of product from the same number of cycles from two different
types of cells, (and standardizing them with products from genes that
are expressed at similar levels in the two cells), an investigator can
determine the relative abundance of mRNA for that particular gene in
the two cell types. This method is at least as quantitative as Northern
blot analysis and RPA, and can be accomplished more quickly and with
smaller amounts of starting material.
Figure 5. Semiquantitativepolymerasechain reaction
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