Expression Profi ling of Components of the miRNA Maturation Machinery - miRNA Maturation: Methods and Protocols - page 63

The primer sequences were determined using the Primer
Express software (Applied Biosystems, USA) and were synthesized
by a custom service provider that specializes in the synthesis of
oligonucleotides ( see Notes 8 and 9 ).
The primers that we have successfully used for amplifi cation
include the following:

Dicer1

Forward 5 ′ -ttaaccttttggtgtttgatgagtgt-3 ′ .

Reverse 5 ′ -ggacatgatggacaattttcaca-3 ′ .

Drosha

Forward 5 ′ -catgtcacagaatgtcgttcca-3 ′ .

Reverse 5 ′ -gggtgaagcagcctcagattt-3 ′ .

PACT

Forward 5 ′ -tgcagttcctgaccccttaatg-3 ′ .

Reverse 5 ′ -agccaattcctgtaatgaaccaa-3 ′ .

DGCR
Forward 5 ′ -gcaagatgcacccacaaaga-3 ′ .

Reverse 5 ′ -ttgaggacacgctgcatgtac-3 ′ .

TARBP1

Forward 5 ′ -cattaatggatgcgctttcaga-3 ′ .

Reverse 5 ′ -tgtaatttcagtcccaatggagaac-3 ′ .

TARBP2

Forward 5 ′ -ggttgccggagtacacagtga-3 ′ .

Reverse 5 ′ -tgccactcccaatctcaatg-3 ′ .

1. PCR mixture:

5 μ l cDNA solution.

●

12.5 l 2× Power SYBR Green PCR Mix (Applied

Biosystems).

μ

●

5 M of each primer and diethylpyrocarbonate (DEPC)-

treated water to a fi nal volume of 25

μ

●

l.
2. The amplifi cation reaction is performed in the StepOne Real-
Time PCR System (Applied Biosystems, USA) using the fol-
lowing temperature protocol:

μ

(a) For PCR master mix activation: 10 min at 95 °C.

(b) PCR: 15 s at 95 °C and 60 s at 60 °C.
Because the SYBR ® Green dye binds both the specifi c and
nonspecifi c DNA, the double-stranded PCR products are
melted at 95 °C after 40 cycles. This process linearly decreases