Biology Reference
In-Depth Information
Chapter 4
Expression Profi ling of Components of the miRNA
Maturation Machinery
Michael Sand and Marina Skrygan
Abstract
The quantitative real-time polymerase chain reaction (qRT-PCR) is a valuable and well-proven technique
used to investigate the expression level of multiple components of the microRNA (miRNA) maturation
machinery. Here, we describe how to determine the messenger RNA expression levels of components of
the miRNA machinery starting from the isolation of the RNA from a tissue biopsy to performance of the
qRT-PCR.
Key words
MicroRNA maturation, Quantitative real-time polymerase chain reaction (qRT-PCR)
1
Introduction
Every protein has a characteristic amino acid sequence, which is
determined by the coding gene. A gene is not immediately trans-
lated into a protein; it is expressed via the intermediate production
of messenger RNA (mRNA). Chemically similar to DNA, RNA is
a straight, high-molecular-weight polymer. mRNA is synthesized
by the same process of complementary base pairing as occurs dur-
ing the replication of DNA; however, mRNA is read from one
strand of the DNA double helix, resulting in a single strand that is
complementary only to that DNA strand. Apart from the substitu-
tion of thymine with uracil, the mRNA strand generated is identi-
cal to the remaining DNA strand. In addition, the mRNA contains
a sequence of nucleotides that is composed of a sequence of exons,
which corresponds to the sequence of amino acids in the protein.
The intron regions of the DNA that do not contain amino acid
coding information are excised during the synthesis of the mRNA.
The transcription of a particular mRNA begins when a cell needs a
particular protein.
