Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 237

sequences flanking each side of the siRNA/miRNA target site were added to enhance
the efficiency of blocking siRNA/miRNA activity ( Hutvagner et al., 2004 ). For
affinity purification of miRNPs, we found that the flanking sequences are not
necessary (our unpublished observation). For example, we used a 5 0 -biotinylated
22-nt 2 0 -O-methyl oligonucleotide complementary to let-7 and a 22-nt oligonucle-
otide complementary to luciferase mRNA sequence as negative control.
3. Affinity Purification
Incubate 300 m L of cell extract with 15 pmol of 5 0 -biotinylated 2 0 -O-methy
oligonucleotide at RT for 1 h. Subsequently, add 20 m L of Streptavidin Sepharose
(GE Healthcare), pre-equilibrated with the homogenization buffer, and incubate at
RT for 1 h with gentle agitation on a nutator. Centrifuge at 8000 rpm for 30 s and
remove the unbound fraction. Wash five times by resuspending with 1 mL of
homogenization buffer and centrifuging at 8000 rpm for 30 s each time. For analysis
of purified proteins, resuspend the beads in a final volume of about 28 m L of water
and add 10 m L XT sample buffer (4 ) and 2 m L XT reducing agent (20 ) (Bio-Rad)
or 2-mercaptoethanol. Heat at 95 C for 5 min. Remove the beads by passing the
sample through a Spin X cellulose acetate filter (Corning Costar). Analyze the eluted
proteins by denaturing gel electrophoresis using a Criterion XT Bis-Tris 4-12%
gradient gel (Bio-Rad) and western blotting with antibodies against proteins of
interest. RNAs in the beads or unbound fraction can be extracted by Trizol and
analyzed by northern blotting.
VI. Construction of Transgenic Strains to Analyze miRNA Expression and
Target Regulation
Northern blotting, real-time quantitative PCR, and deep sequencing are powerful
tools for analysis of miRNAs in C. elegans, but they lack the ability to detect
spatiotemporal patterns of expression. Reporter genes, such as gfp (encoding green
fluorescent protein) ( Chalfie et al., 1994 )orlacZ (encoding beta-galatosidase)
( Fire et al., 1990 ), driven by a miRNA promoter (Pmir) are more amenable to
analyze spatiotemporal miRNA expression in vivo. Moreover, a series of deletions
in the promoter region of the construct can be used to determine the transcriptional
regulatory cis-elements of miRNAs. For example, our group has examined the
expression pattern of lin-4 and let-7 family members using Pmir::gfp constructs
( Esquela-Kerscher et al., 2005; Johnson et al., 2005 ). In addition, by serially deleting
the let-7 promoter region upstream of the gfp reporter, we have succeeded in
pinpointing a short temporal regulatory cis-element (TRE) of let-7 ( Johnson et al.,
2003 ). We also have demonstrated that hbl-1, one target of let-7, inhibits the tran-
scription of let-7 using a Plet-7::gfp construct in which the putative HBL-1 binding
sites were deleted ( Roush and Slack, 2009 ). Recently, Martinez et al. (2008) have
used Pmir::gfp reporter constructs to analyze spatiotemporal promoter activity of
miRNA on a genomic scale ( Martinez et al., 2008 ). With all its promises, one
obvious limit of the miRNA promoter-reporter fusion approach is that it can only