Biology Reference
In-Depth Information
The microRNA (miRNA) maturation machinery is composed
of a well-defi ned set of proteins including the intranuclear RNase
III endonuclease Drosha and its cofactor DiGeorge syndrome
critical region gene 8 (DGCR8 or Pasha), the miRNA nuclear
export receptor RAN GTPase exportin-5 (Exp5), and the RNase
III enzyme Dicer and its cofactors the double-stranded RNA-
binding domain (dsRBD) protein Tar RNA-binding protein
(TRBP) and the protein activator of PKR (PACT). Recently, a
number of studies have provided growing evidence for a role of the
miRNA maturation machinery in a variety of diseases, suggesting
that it could serve as a prognostic tool [
1
-
10
]. In particular, the
miRNA maturation machinery has been investigated in a variety of
different tumors.
In this chapter, we share our experience of the expression profi l-
ing of components of the miRNA maturation machinery in the skin,
which can be analogously performed with other tissue [
11
,
12
].
2
Materials
1. RNA
later
®
RNA Stabilization Reagent (QIAGEN).
2. RNeasy
®
Lipid Tissue Mini Kit (QIAGEN).
3. Stainless steel beads (5 mm).
4. Chloroform (99 %, molecular biology grade).
5. Isopropanol (99.7 % analytical grade).
6. DNAse I (RNAse free, 1,500 U).
7. DNase I (10 U/
l; RNase free).
8. TaqMan
®
Reverse Transcription Reagents Kit (Applied
Biosystems).
9.
Power
SYBR Green PCR Mix (Applied Biosystems).
10. Gel-loading buffer.
11. Agarose HR-Plus.
12. NuSieve agarose.
13. 50× TAE buffer (commercial).
14. 100-bp DNA ladder.
15. 1 % ethidium bromide solution.
μ
3
Methods
3.1 Isolation of Total
RNA from a Tissue
Biopsy
R NAlater-fi xed tissue can be stored for 1 day at 37 °C, for 7 days
at 15-25 °C, or 4 weeks at 2-8 °C (
see
Notes 1
and
2
). For longer
storage periods, the fi xed tissue should be maintained at −20 or
−80 °C. Total cellular RNA is isolated using the RNeasy
®
Lipid
