Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 228

H. Small RNA Purification
Prepare 10 m g of total RNA for each experimental condition (see Section I for total
RNA isolation). The integrity of RNA can be evaluated by separating RNA on a 1%
agarose gel and EtBr staining. A high-quality RNA sample should show two discrete
bands at 3.5 kb and 1.8 kb that represent 26S rRNA and 18S rRNA, respectively.
Separate 10 m g of total RNA by electrophoresis on a 15% TBE-urea gel along with
20-100 bases ladder that should be loaded several lanes away from the total RNA
sample. Stain the gel with EtBr and view the gel on a Dark Reader transilluminator
(Clare Chemical Research) or a long wavelength UV transilluminator. Cut out a band
of gel in the sample lane corresponding to the 18-30 nt bands in the marker lane.
Elute and precipitate RNA from the gel slice (see Section III, Modified RACE to
map cleavage sites). Resuspend the RNA pellet in ultrapure water.
I. cDNA Library Preparation
In order to prepare the cDNA libraries, RNA adaptors for 5 0 - and 3 0 -ends are
ligated to the purified small RNAs for use in reverse transcription and PCR ampli-
fication. The 3 0 -adaptor also possesses a sequence that is in the reverse complemen-
tary orientation to a surface-bound amplification primer on the Illumina Genome
Analyzer flowcell. The sequences of another flowcell primer and the sequencing
primer will be added to the other end of the template by a PCR primer. The adaptors
must be designed to take advantage of the 5 0 -phosphate and 3 0 -hydroxyl termini of
small RNAs, resulting from RNase III processing. In contrast, most RNase degra-
dation products have 5 0 -hydroxyl and 3 0 -phosphate termini. The 5 0 -adaptor should
carry a hydroxyl group at the 3 0 -terminus as an acceptor for 5 0 -phosphate containing
small RNAs, while the 3 0 -adaptor should carry a 5 0 -phosphate terminus as a donor
and a non-nucleotidic moiety at the 3 0 -terminus to prevent circularization. Use
excess amounts of adaptors over 5 0 -phosphate containing small RNAs to avoid
circularization. Alternatively, use a 3 0 -adaptor with a preadenylated 5 0 -end and a
truncated T4 RNA ligase 2, RNL2(1-249), in the absence of ATP to reduce back-
ground ligation ( Hafner et al., 2008 ). Perform reverse transcription over the ligated
RNA products and then amplify by PCR. Reagents, adaptors, primers, and detailed
protocols are available from Illumina, Inc.
1. Adaptor Ligation
Dissolve purified small RNA in 5.7 m L of ultrapure water, and mix with 1.3 m Lof
5 0 -RNA adaptor (Illumina part #1000595), 1 m Lof10 T4 RNA ligase buffer, 1 m L
of RNaseOUTand 1 m L of T4 RNA ligase. Incubate the reaction mixture at 20 C for
6 h in a thermal cycler and hold at 4 C. Separate ligated RNAs by electrophoresis on
a 15% TBE-urea gel along with 20-100 bases ladder and recover RNAs correspond-
ing to 40-60 nt bands using the same elution and ethanol precipitation steps men-
tioned above. Dissolve purified small RNA in 6.4 m L of ultrapure water, and mix