Supplementary information
Materials and Methods
RT-PCR
One-step RT-PCR (Invitrogen) was used to obtain the fragments of the full-length of
DP100. Total RNAs extracted from CT26 tumors were used as templates. Forward
and reverse primers were designed according to the sequences of mouse genome
contig NT_082868, as discussed in the Supplementary results section. Reverse
transcription was performed at 50C for 30 min. After incubation at 94C to
inactivate reverse transcriptases and activate DNA polymerase, PCR reactions were
followed. To address whether the RT-PCR products were due to contamination of
genomic DNA, control reactions were performed where the reaction mixtures were
held at 4C at the stage of reverse transcription (RT) so that no RT occurred, and
processed through all the other steps for RT-PCR. One-step RT-PCR products were
sequenced by the institutional sequencing core facility using ABI 377 DNA
sequencers. Aliquots of the RT-PCR products were resolved on 1% agarose gels.
5'- and 3'-RACE
GeneRacer (Invitrogen) using RNA ligase-mediated rapid amplification from the 5'
and 3' ends (RLM-RACE) was applied to reach the 5' and 3' ends of the full-length
DP100. In brief, 5 µg of total RNA from CT26 tumor was treated as described by the
manufacturer and ligated to the GeneRacer RNA oligo. The primers used to produce
the first strand cDNAs were either the Oligo dT GeneRacer primer or a reverse primer
specific for the DP100 sequence (primer RACE100_AS: 5'-AAC GTA CAT GCG
s1
CCA GTC GAA GGA-3'). For the 5'-RACE, cDNAs transcribed with the OligodT
primer or the RACE100_AS primer were used as the templates, the GeneRacer 5'
primer (G5) served as the forward primer, and either the RACE100_AS primer or a
primer corresponding to more to the 5' end of DP100 sequence (primer DP100_AS:
5'-TCT CCT AAA CTG CTC TGG TCA GCC TCC ATT A-3') was used as the
reverse primer. For the 3'-RACE, cDNAs transcribed with the Oligo dT primer were
used as templates, the GeneRacer 3' (G3) primer served as the reverse primer, and a
gene specific primer for the extended sequence of DP100 corresponding to more to
the 3' end (primer ext6720: 5'-TTC AAG TCC CTG CGG TGT CTT TG-3') was used
as the forward primer. Aliquots of RACE products were resolved on 1% agarose gels.
Results
Identification of the full-length of hcn gene
5’ RACE of DP100 RNA was used to extend to the 5' end of the full-length hcn gene
(Figure S1a). The hcn sequence was identical to part of the sequence of only one
genomic contig from the NCBI mouse genome database, namely NT_082868.
BLAST search revealed numerous EST hits matched sequentially to the sequence of
NT_082868 starting from the nucleotide corresponding to the 5’ end and extending
~7-kb downstream (data not shown). Proposing that the 7-kb hcn gene was within
this region, 4 sets of primers were designed to amplify the additional segments of this
region. Products were generated in all four sets of RT-PCR reactions (Figure S1b,
lanes 1, 4, 7, and 10). Sequences of all four RT-PCR products were assembled into a
6850 bp fragment. Finally, 3'-RACE was used to reach to the 3' end of the HCN
transcript. A DNA band of ~300 bp was observed (Figure S1c). Sequencing of the
s2
DNA revealed the authentic sequence of NT_082868 followed by a stretch of 24 As at
the 3' end. Because the 24 A segment was not present in the genomic sequence at this
position in NT_082868, we concluded that the poly A tail indicated the 3' end of the
HCN transcript.
Table
Table S1 In situ hybridization analysis of MALAT-1 in various human carcinomas.
Breast Cancer
10
Numberb (%)
with
Elevated HCN
8 (80)
Pancreas Cancer
12
7 (58)
0
Lung Cancer
32
16 (50)
0
Colon Cancer
18
9 (50)
0
Prostate Cancer
32
7 (22)
0
Cancer Type
Numbera
Analyzed
Numberc
with
Decreased HCN
0
a
Number of tumor/non-tumor pairs from different human patients in the tissue
microarray slides. b,cThe intensity of MALAT-1 staining was scored as undetectable or
low (0), moderate (1), strong (2), or very strong (3). The numbers represent tumor
samples with higher b or lower c scores than the corresponding non-tumor pairs.
s3
Figure legend
Figure S1 Identification of the full-length of hcn gene. (a) Identification of the 5’end (5’-RACE), (b) middle section (RT-PCRs), and (c) 3’-end (3’-RACE) of the fulllength DP100 transcript. For the 5’-RACE, the DP100_AS (for lanes 1 and 3) or the
RACE_100AS (lanes 2 and 4) were used as reverse primers, and the first strand
cDNAs were generated with either the OligodT primer (lanes 1 and 2) or the
RACE100_AS primer (lanes 3 and 4). For the RT-PCRs, primers were generated to
amplify nt 1-2170 (I), nt 1290-3830 (II), nt 3740-5830 (III), and nt (5600-6850) of the
proposed full-length DP100 gene. RT, reverse transcription; DNA, genomic DNA
from CT26 tumors.
s4