Processes 2018, 6,42
2.4. Small RNA-Seq Analysis
To generate miRNA expressions data, reads from the small RNA-Seq experiment were aligned to
known miRNA transcripts using the BLAST+ [39] sequence aligner. In order to be reported as valid
the alignment was required to be over 99% similar with no more than one mismatch over 36 base pairs.
2.5. Cell Growth Assay
MDA-MB-231 cells were first transfected with either 100 nmol/L of miR-140 antagomir (Anti-140)
(Cat # C-301055-01-0005, GE Healthcare Dharmacon, Chicago, IL, USA) or scrambled negative control
(Ctrl-140) (catalog number CN-001000-01-05, Dharmacon) using Lipofectamine (Life Technologies,
Carlsbad, CA, USA) according to the manufacturers protocol. Cell number was determined by trypan
blue staining and manual counting at 24, 36, and 48 h post-transfection. Growth was determined as
the relative cell number compared with vehicle-treated (0.1% DMSO) controls.
2.6. Western Blot Analysis
Following transfection of cells with anti-miRNAs, at 36 h existing media was replaced with lysis
buffer containing protease inhibitors, incubated for 15 min at 4 ◦ C, and then transferred to tubes.
The cell proteins were electrophoresed through an 8% SDS–polyacrylamide gel and transferred to
polyvinylidene fluoride membranes for the immobilization of the proteins. The membranes were
blocked for 1 h in 2% non-fat milk in phosphate-buffered saline containing 0.05% Tween-20 surfactant
and then washed and incubated with primary DFFA (ICAD) antibody (LF-PA0058, Thermo Scientific,
Rockford, IL, USA) overnight at 4 ◦ C. Following subsequent washing and incubation with goat
anti-rabbit peroxidase-conjugated secondary antibody the immunoreactive bands were visualized and
quantified using a Flurochem densitometer for the reporting of the protein levels.
3. Results
In order to characterize transcriptional differences between MCF-7 and MDA-MB-231 cells, RNA
was isolated from each and split into “mRNA” (>200 nt RNAs) and “small RNA” (<200 nt RNAs
including mature miRNAs) fractions. These samples were commercially sequenced resulting in over
2 billion nucleotides of small RNA reads and roughly 6 billion nucleotides of the longer mRNA reads.
3.1. Identification of RNA Edit Sites
Identification of putative RNA edit sites within each of the two cell lines was performed by
mapping RNA-Seq reads to the GRCh38 human reference genome. As read alignments are reported
with respect to the leading strand of the reference genome, a putative edit site would appear as an
A-to-G mutation if the read arose from the forward strand, or a T-to-C mutation in the case of the
reverse strand (Figure 3). In all, 19,462 unique edit sites were identified in MCF-7 and 35,090 sites
were found in MDA-MB-231 (Table S1). That said, we found reads containing edits differed from the
reference genome at the edit position 51.8% of the time in MCF-7s on average and 49.8% of the time
in MDA-MB-231s.
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2.4. Small RNA-Seq Analysis
To generate miRNA expressions data, reads from the small RNA-Seq experiment were aligned to
known miRNA transcripts using the BLAST+ [39] sequence aligner. In order to be reported as valid
the alignment was required to be over 99% similar with no more than one mismatch over 36 base pairs.
2.5. Cell Growth Assay
MDA-MB-231 cells were first transfected with either 100 nmol/L of miR-140 antagomir (Anti-140)
(Cat # C-301055-01-0005, GE Healthcare Dharmacon, Chicago, IL, USA) or scrambled negative control
(Ctrl-140) (catalog number CN-001000-01-05, Dharmacon) using Lipofectamine (Life Technologies,
Carlsbad, CA, USA) according to the manufacturers protocol. Cell number was determined by trypan
blue staining and manual counting at 24, 36, and 48 h post-transfection. Growth was determined as
the relative cell number compared with vehicle-treated (0.1% DMSO) controls.
2.6. Western Blot Analysis
Following transfection of cells with anti-miRNAs, at 36 h existing media was replaced with lysis
buffer containing protease inhibitors, incubated for 15 min at 4 ◦ C, and then transferred to tubes.
The cell proteins were electrophoresed through an 8% SDS–polyacrylamide gel and transferred to
polyvinylidene fluoride membranes for the immobilization of the proteins. The membranes were
blocked for 1 h in 2% non-fat milk in phosphate-buffered saline containing 0.05% Tween-20 surfactant
and then washed and incubated with primary DFFA (ICAD) antibody (LF-PA0058, Thermo Scientific,
Rockford, IL, USA) overnight at 4 ◦ C. Following subsequent washing and incubation with goat
anti-rabbit peroxidase-conjugated secondary antibody the immunoreactive bands were visualized and
quantified using a Flurochem densitometer for the reporting of the protein levels.
3. Results
In order to characterize transcriptional differences between MCF-7 and MDA-MB-231 cells, RNA
was isolated from each and split into “mRNA” (>200 nt RNAs) and “small RNA” (<200 nt RNAs
including mature miRNAs) fractions. These samples were commercially sequenced resulting in over
2 billion nucleotides of small RNA reads and roughly 6 billion nucleotides of the longer mRNA reads.
3.1. Identification of RNA Edit Sites
Identification of putative RNA edit sites within each of the two cell lines was performed by
mapping RNA-Seq reads to the GRCh38 human reference genome. As read alignments are reported
with respect to the leading strand of the reference genome, a putative edit site would appear as an
A-to-G mutation if the read arose from the forward strand, or a T-to-C mutation in the case of the
reverse strand (Figure 3). In all, 19,462 unique edit sites were identified in MCF-7 and 35,090 sites
were found in MDA-MB-231 (Table S1). That said, we found reads containing edits differed from the
reference genome at the edit position 51.8% of the time in MCF-7s on average and 49.8% of the time
in MDA-MB-231s.
134