Expression Profi ling of Components of the miRNA Maturation Machinery - miRNA Maturation: Methods and Protocols - page 64

Fig. 2 Dissociation (melting) curve of the PCR products

the fl uorescence intensity of the dye until it reaches the melting
temperature, which is specifi c for the DNA target sequence.
For example, the melting temperature for Dicer1 is 76 °C.
When the melting temperature is reached, the DNA fragment
denatures into a single strand and releases the entire fl uores-
cent dye. The specifi c melting temperature depends on the size
of the PCR amplifi cate; therefore, the nonspecifi c products and
primer dimers melt at different temperatures ( see Note 10) . A
melting or a dissociation curve specifi cally detects the amplifi ed
target products (Fig. 2 ). In addition, the amplifi cate size is
monitored using agarose gel electrophoresis.
3. The housekeeping genes (HKGs) GAPDH and RPL38 were
investigated for their expression stability. The expression level
of RPL38 was shown to be the least regulated of all the HKGs
studied; therefore, RPL38 was used as the reference gene for
the relative quantifi cation of the PCR products ( see Note 11 ).
RPL 38

Forward 5 ′ -TCACTGACAAAGAGAAGGCAGAGA-3 ′ .

Reverse 5 ′ -TCAGTGTGTCTGGTTCATTTCAGTT-3 ′ .