Supplemental Data for:
Baskerville and Bartel, RNA 11, pp. 241-247
Supplemental Material
Figure S1. (A)
Pilot microarray. miRNA probes were printed to test hybridization and wash conditions.
Samples for hybridization to the array were individually labeled with either
(Cy3, green) or (Cy5, red), mixed in equamolar amounts, and hybridized to the
array at the temperatures indicated. Specific hybridization was observed as
either red or green spots on the array, whereas crosshybridization was observed
as shades of yellow. Each miRNA probe was selected to test the resolution of
the array for single and multiple mismatches between probe sequences. For example,
fug-mir-197 and hsa-mir-197 test the resolution of single nucleotide differences
within three nucleotides of the 3_ end of the probe sequence. Test probes were
also designed to span a range of melting temperatures. As the hybridization
temperature was increased, the specificity of the test array was generally improved
to a point near 55-60º C (see hsa-mir-148 and hsa-mir-136). (B) Representative
sample grid of a microarray. A more extensive microarray was printed with probes
covering a set of non-redundant human and mouse miRNA sequences. As a test of
the array specificity, the oligonucleotides used to construct the reference
sample were split into two different sets and labeled as described above, mixed
and hybridized to the array at 57º C. This subgrid contains replicates samples
of individual probes (top four rows and bottom four rows); test samples reproducibly
hybridize within the array, and little cross-hybridization was observed, although
it is visible in some cases (yellow spots in subgrid shown).
Figure S2. Correlation
between cloning frequency and microarray score.
Small RNAs sequences were amplified from total RNA isolated from C. elegans
mixed-stage worms and then dye-labeled for hybridization to the array. Microarray
scores for mixed-stage C. elegans samples were compared with published cloning
frequencies from the same samples. In general, cloning frequency and chip score
are positively correlated (R2. = 0.66), similar to the results comparing cloning
frequency to molecular abundance (R2 = 0.78) (Lau et al., 2001).
Figure S3. Biological
replicates of microarray data. Microarray sample libraries were prepared from
primary human hepatocytes enriched from two separate transplant-grade livers.
Each library was cloned independently and hybridized to the array. The reproducibility
of the array data between these samples (R2 = 0.93) suggests that the profile
of miRNA expression within organs is consistent and reproducible, as are the
methods for amplifying and labeling the miRNA sequences.
Supplemental Table 1. Microarray Data
Supplemental Table 2. Microarray Probe Sequences