Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 226

amplify cDNA with a gene-specific forward primer and a reverse primer comple-
mentary to the 3 0 linker. Use PCR conditions appropriate for the primer melting
temperatures and expected product sizes with 30 cycles of amplification in a 50 m L
reaction using High Fidelity Taq (Invitrogen). Clean the PCR reaction using the
Qiagen PCR column purification system and elute in 50 m L of ddH 2 O. Use 1 m Lof
the first PCR reaction to perform a nested PCR, using a second set of primers
corresponding to a gene-specific nested forward primer and a linker specific reverse
nested primer. To identify specific PCR products, also set up nested PCR reactions
that contain the gene-specific and 3 0 nested primers individually. Use appropriate
PCR conditions with 25-30 cycles of amplification in a 50 m L reaction using High
Fidelity Taq (Invitrogen). Clean the reactions as described above and use half of the
reaction (25 m L) to analyze the PCR products by separation in an agarose gel.
Specific products should be present in the nested PCR using both primers but not
in the control reactions using the single primers. If multiple products are present,
perform gel purification of the specific bands before cloning. Otherwise, the PCR
reaction can be used directly for cloning using the TOPO reagents (Invitrogen).
Cloned DNA is transformed into bacteria, cultured, and the plasmids isolated by
standard methods. Restriction digestion analyses should be used to verify inserts
before subjecting clones for sequencing and identification of the 3 0 cleavage site.
2. Characterize the 5 0 End of 3 0 Drosha Cleavage Products
Incubate GeneRacer RNA Oligo with 1-5 m g of total RNA, isolated as described
above in Section I, at 65 C for 5 min and 4 C for 2 min. Add 40 U of RNaseOut, 5 U
of T4 RNA ligase (Fermentas), 1 m L of 10 mM ATP, and 1 m Lof10 T4 ligase
buffer (Fermentas). Incubate at 37 C for 1 h before extracting RNA as described in
the second RNA-extraction step of Section I. Resuspend ligated RNA in 10 m Lof
DEPC water after precipitation. Reverse transcribe as described above with 1 m Lof
50 m Mgene-specific reverse primer. Amplify cDNAwith GeneRacer 5 0 primer and a
gene-specific reverse primer as described above. Clean the PCR reaction and per-
form a nested PCR with the GeneRacer 5 0 nested primer and a gene-specific reverse
nested primer as described above. Analyze the nested PCR reaction, TOPO clone,
and sequence as described above to identify the 5 0 cleavage site.
IV. Deep Sequencing of Small RNA Populations
High-throughput ''deep sequencing'' technologies, such as 454 Life Science/
Roche Genome Sequencer ( Margulies et al., 2005 ), Solexa/Illumina Genome
Analyzer ( Bentley et al., 2008 ), and Applied Biosystems SOLiD System (for review
see Mardis, 2008; Shendure and Ji, 2008 ), capable of simultaneously sequencing
millions of molecules, have provided an excellent tool for small RNA profiling and
discovery. Deep sequencing outperforms other existing profiling methods, such as
array hybridization or qRT-PCR, in discriminating closely related RNAs, detecting
5 0 -or3 0 -end variations and measuring absolute abundance with a better dynamic
range. Furthermore, deep sequencing, unlike microarrays, allows the identification