Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 221

loading and nondegraded RNA samples. Equilibrate membrane (Zeta-Probe GT,
Bio-Rad) cut to the same size as the gel in water followed by 0.5 TBE. Assemble
transfer apparatus by layering: plastic holder, sponge pad wetted in 0.5 TBE, two
sheets of Whatman 3MMpaper cut to the size of the gel and wetted in 0.5 TBE, gel,
membrane, two sheets of Whatman 3MM paper prepared as before, sponge pad
wetted in 0.5 TBE, and the plastic holder. Transfer at 200 mA at 4 C for 2-4 h. In
our experience, wet transfer methods are much more reliable than semidry dry
methods for transfer of small RNAs for northern blotting analyses.

2. Prehybridization, Probe Preparation, and Hybridization
Place membrane RNA side up on a piece of dry Whatman 3MM paper and cross-
link at 1200 m J 100 by using a Spectrolinker SL-1000 UV Crosslinker. Sandwich
membrane between dry Whatman 3MM paper and bake in an oven at 80 C for
30 min. Depending on the miRNA GC content, incubate blot at 40-50 C with
agitation for at least 2 h in a sealed bag containing 25 mL prehybridization solution.
Multiple methods can be used to make a radioactive probe to target the mature
miRNA of interest. A simple, common method is based on the microRNA Starfire
Kit (IDT DNA) that requires an oligo complimentary to the miRNA of interest and
containing the proprietary starfire sequence (IDT DNA) and 80 m Ci of alpha- 32 P
dATP (6000 Ci/mmol). For more abundant miRNAs, a probe can be made by
labeling a DNA oligo complementary to the miRNA of interest. To do this incubate
25 pmol of the DNA oligo with g

32

PATP (6000 Ci/mmol), T4 polynucleotide kinase
(PNK) buffer, and T4 PNK enzyme at 37 C for 1 h. Add the probe to 25 mL of fresh
prehybridization solution. Remove the original prehybridization solution from the
blot container before adding the fresh probe solution. Incubate the blot in probe
solution at 40-50 C with agitation for at least 4 h or up to overnight.

3. Washing, Viewing, and Stripping
Probe solution can be stored at 20 C and reused. Blots should be washed,
viewed, and stripped as described above for agarose northern blotting.

E. Analysis of Primary, Precursor, and Mature miRNAs by qRT-PCR
This analysis allows detection of miRNAs from a small amount of starting mate-
rial, but does not distinguish between differently sized primary miRNA isoforms, or
determine the origin of pre-miRNA since pre-miRNA is contained within pri-
miRNA ( Bracht et al., 2010; Van Wynsberghe et al., 2011 ). Inclusion of a control
sample that lacks reverse transcriptase will test if any amplification is caused by
genomic DNA. SYBR Green or Taqman qRT-PCR can be performed. Taqman qRT-
PCR is more specific than SYBR Green qRT-PCR because of the additional probe,
but it is also more costly. qRT-PCR can be performed in one step or in two steps of