Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 219

cytoplasm releases the 22 nt mature miRNA ( Davis and Hata, 2009 ). Regulation of
mature miRNA levels can be accomplished by controlling any step in the miRNA
biogenesis pathway ( Winter et al., 2009 ). Though some control mechanisms ubiq-
uitously affect miRNA expression, others are specific for particular miRNAs ( Kai
and Pasquinelli, 2010; Winter et al., 2009 ). To gain a complete understanding of
miRNA expression and determine the mechanisms controlling miRNA biogenesis,
analysis of pri-, pre-, and mature miRNA levels is necessary.
C. Analysis of Primary miRNAs by Agarose Northern Blotting
This method allows concurrent analysis of multiple pri-miRNA isoforms, depend-
ing on sufficient differences in their sizes. For example, the three pri-let-7 transcripts
or two pri-lin-4 transcripts in C. elegans can be resolved by denaturing 1% agarose
gels and detected by this northern blotting protocol ( Bracht et al., 2004; Bracht et al.,
2010; Van Wynsberghe et al., 2011 ). The northern blotting protocol is comparable to
that used to detect mRNAs except that the often low abundance of pri-miRNAs
necessitates that at least 10 m g of total RNA, prepared by the method described above
in Section I, is used for each sample.
1. Running and Transferring
Make a 1% agarose gel in MOPS/EDTA running buffer (40 mM MOPS pH 7.0,
2 mM EDTA). Prepare samples by adding 10 m g of total RNA in a total volume of
10 m Lto15 m L of MOPS/EDTA running buffer and 5 m L of 37% formaldehyde. Heat
samples at 70 C for 10min and then transfer to ice for 1min. Add 6 m Lof6 glycerol
loading buffer (30% glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol) to each
sample before loading. Run the gel at 80 to 125 V for at least 2 h. Visualization of the
rRNA bands by ethidium bromide (EtBr) staining is used to confirm even loading and
nondegraded RNA samples before proceeding with northern blotting. Rinse gel gently
in sterile water with shaking. Equilibrate membrane (Bio-Rad Zeta Probe GT), cut to
the same size as the gel, in ddH 2 Ofollowedby10 SSC (3 M NaCl, 0.3 M sodium
citrate, pH 7.0). Assemble the transferring setup at RT by layering: glass tray contain-
ing 10 SSC, upside-down gel casting tray, two sheets of Whatman 3MM paper
wetted in 10 SSCwithendsdippedinthe10 SSC in the glass tray to act as wicks,
gel with bottom side facing up, membrane, three pieces of Whatman 3MM paper
wetted in 10 SSC and cut to the size of the membrane, a stack of paper towels 5-10
cm high and cut to the size of the gel, and a light weight evenly stacked on top. Allow
transfer to occur at least 6 h and up to overnight.
2. Prehybridization, Probe Preparation, and Hybridization
Briefly rinse the membrane in 6 SSC to remove any agarose particles. Place the
side of the membrane that contacted the gel facing up on a piece of dry Whatman
3MM paper and cross-link at 1200 m J 100 by using a Spectrolinker XL-1000 UV