Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 217

Originally discovered in C. elegans as genes that regulate developmental timing,
miRNAs regulate fundamental processes in diverse organisms and are often con-
served between species ( Lee et al., 1993; Pasquinelli et al., 2000; Reinhart et al.,
2000 ). This conservation, as well as the availability of miRNA mutants makes
C. elegans an important system to study the mechanisms of miRNA biogenesis,
target identification, and function ( Kato and Slack, 2008 ).
This chapter details current methods used to isolate miRNA species, analyze
primary, precursor, and mature miRNA expression, map the 5 0 and 3 0 ends of
miRNA primary and cleavage products, identify small RNA populations, analyze
miRNA-protein complexes, and construct reporters and transgenic strains to
analyze miRNA target regulation. Though specific for the analysis of small RNAs,
these methods can also be adapted to study mRNA expression or mRNA-protein
interactions.
II. Rationale, Methods, and Materials
I. Total RNA isolation
II. Analysis of primary, precursor, and mature miRNA expression
III. RACE mapping of miRNA primary and cleavage products
IV. Deep sequencing of small RNA populations
V. Analysis of miRNP complexes
VI. Construction of transgenic strains to analyze miRNA expression and target
regulation
I. Total RNA Isolation
Though standard protocols can be used for RNA isolation in C. elegans, to ensure
isolation of high-quality RNAs of all sizes, including small 22 nt mature miRNAs,
the following double-RNA-extraction method is preferred. This method is based
upon the Trizol RNA isolation protocol (Invitrogen) and can be used to isolate RNA
from embryos or larval and adult stage worms. RNA generated from this procedure
can be used for any application, including those described in Sections II to V.
A. Worm Collection and Preparation for RNA Isolation
For analysis of miRNA expression at specific developmental time points, plate
synchronized larval stage 1 (L1) worms on the appropriate bacterial food source and
wash worms off plates with M9 at the desired time point. Rock worms inM9 (22 mM
KH 2 PO 4 ,42mMNa 2 HPO 4 , 85.5 mM NaCl, 1 mMMgSO 4 ) in 15 mL conical tubes
for 20 min to allow digestion of bacterial food. Pellet worms with centrifugation and
transfer to 1.5 mL tubes. Pellet again, remove excess liquid, and snap-freeze in a dry
ice-ethanol bath. Worm pellets of 50-100 m L will yield at least 50 m g of total RNA.