Yohei Miyagi
Supplementary Information
Materials and Methods
Reversetranscriptase-polymerase chain reaction (RT-PCR)
RNA samples were first subjected to primary RT-PCR using 200 ng of sample RNA with the
SuperScript III One-Step RT-PCR system (Invitrogen, Carlsbad, CA) according to the
manufacturer’s instruction. Briefly, the reaction mixture contained 200 ng of sample RNA, 5
pmoles
of
each
forward
and
reverse
PCR
primer,
and
the
SuperScript
III
reversetranscriptase/Platinum taq polymerase mixture provided by the manufacturer in 25 µl of the
recommended buffer. The reaction consisted of incubation at 55 ºC for 30 min for
reversetranscriptation followed by 42 cycles of 94 ºC for 20 sec, 58 ºC for 30 sec and 68 ºC for 30
sec. Then 1.5 µl of the primary reaction was subjected to secondary semi-nested PCR in 25 µl of
the recommended buffer containing 5 pmoles of dNTPs, 5 pmoles of each forward and reverse
PCR primer and 2 units of Platinum taq polymerase (Invitrogen). The reaction consisted of
incubation at 94 ºC for 2 min followed by 28 cycles of 94 ºC for 30 sec, 58 ºC for 30 seconds and 72
ºC for 30 seconds. Five microliters of the amplified reaction was electrophoresed on a 2.5 %
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Yohei Miyagi
agarose gel followed by staining with ethidium bromide and imaging. Primer sequences are
provided in Supplementary Table 1.
5’-Rapid Amplification of cDNA Ends (RACE) Analyses
Total RNA was extracted with TRIzol reagent (Invitrogen). First, expression of the ERG, ETV1,
ETV4 and ETV-5 transcripts and the control porphobilinogen deaminase (PBGD) was evaluated by
conventional RT-PCR with primers located in exons not involved in reported prostate cancer gene
fusions (listed in Supplementary Table 2). Then, 3 µg of each total RNA was subjected to 5’-RACE
with a GeneRacer kit (Invitrogen) according to the manufacturer’s protocol. Briefly, total RNA was
first dephosphorylated with calf intestinal alkaline phosphatase to eliminate truncated RNAs and
non-mRNAs followed by dephosphorylation with tobacco acid pyrophosphatase to remove the cap
structure from the 5’ ends of full-length mRNAs. Oligo RNA of a known sequence (provided in the
kit) was then added to the 5’ end of the RNA with RNA ligase. The ligated RNA was
reversetranscribed to cDNA with random hexamer-primer and used as templates for PCR
amplifications. Amplified products were subcloned into pGem-T-easy plasmid vector (Promega)
and their nucleotide sequences were determined. For RACE of ETV5, the primary RACE PCR
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Yohei Miyagi
gave no products and the nested PCR was performed with the primers given in Supplementary
Table 2.
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