Biology Reference
In-Depth Information
4. Two-Step qRT-PCR
Add 2.5 to 5 m g of total, DNase-treated RNA to 30 pmol of oligo dT or random
primers and 1 m Lof10mMdNTPina12 m L reaction. Incubate at 65 Cfor5min
followed by ice for 1 min. Add 4 m Lof5 buffer, 2 m L of 0.1 M DTT, and 40 U of
RNasin (Promega). Incubate at 42 C for 2 min before adding 200 U of Superscript III
Reverse Transcriptase (Invitrogen). Incubate at 42 Cfor50minand70 C for 15 min.
Add 2 U of RNaseH (Invitrogen), and incubate at 37 C for 30 min. Dilute an aliquot of
the cDNA reaction 1:5 for use in qPCR. As mentioned above the amount of cDNA and
primers used in this reaction may need to be optimized depending on the abundance
of the particular transcript being analyzed. In general, 1 m L of cDNA diluted 1:5 and
6.25 pmol of both forward and reverse primers are added to a minimum 20 m Lreaction
volume containing 1 SYBR Green PCR Mastermix and the Taqman probe if
performing a Taqman reaction (Applied Biosystems). Increasing the reaction volume
will decrease pipetting error but increase the cost per reaction. Reactions are run in a
qPCR thermocycler set to perform the following protocol: (1) 2 min at 48 C, (2) 10min
at 95 C,(3)40cyclesof15sat95 C and 1 min at 60 C, (4) hold at 4 C.
III. RACE Mapping of miRNA Primary and Cleavage Products
Mature miRNAs originate from genome-encoded primary miRNA transcripts that
are often multiple kilobases in length ( Davis and Hata, 2009 ). Primary miRNA
transcripts can be encoded within an intron in the same or opposite direction as its
host gene, or transcribed independently of protein-coding genes ( Davis and Hata,
2009 ). Some miRNAs are clustered together within the same primary miRNA
transcript ( Davis and Hata, 2009 ). Identification of primary miRNA transcripts
defines miRNA clusters, allows further characterization of primary miRNA expres-
sion patterns both through proper promoter-fusion reporter analysis, as described
below in Section VI, and primary miRNA analysis as described above in Section II.
Identification of primary miRNA transcripts also enhances identification of cis and
trans transcription elements. Here we describe RACE (Random Amplification of
cDNA Ends) methods for the identification of 5 0 and 3 0 primary miRNA transcript
ends ( Bracht et al., 2004; Bracht et al., 2010 ).
Similar RACE methods can also be used to identify and characterize cleavage
products of Drosha processing ( Bracht et al., 2010; Van Wynsberghe et al., 2011 ). In
doing so, this method provides a more sensitive method than northern blotting to
determine if precursor miRNA is produced. This method identifies the site of Drosha
processing, which can vary throughout development. Additionally, this method
determines if 3 0 end modification, like uridylation, of precursor miRNA occurs.
5 0 or 3 0 Drosha cleavage fragments destined for degradation can also be identified.
F. 5 0 and 3 0 RACE to Identify Ends of Primary Transcripts
This method is adapted from the GeneRacer Kit (Invitrogen) and requires 1-5 m g
RNA isolated as described above in Section I. Dephosphorylate RNA by incubating
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