Supplemental Materials and Methods
RNA isolation and T-UCR expression microarray analysis
Briefly, 5 g of RNA from each sample was labeled with biotin by reverse
transcription using random octomers. Hybridization was carried out on the second
version of our miRNA-chip (MDACC miRNA Expression Bioarray 19k v5).
Hybridization signals were detected by biotin binding of a Streptavidin-Alexa647
conjugate (one-color signal) using a GenePix 4000B scanner (Axon Instruments).
Images were quantified using the Gene-Pix Pro 6.0 (Axon Instruments). Raw data were
analyzed in BRB-ArrayTools (Biometric Research Branch, National Cancer Institute,
Bethesda, MD, USA, http://linus.nci.nih.gov/BRB-ArrayTools.html). Expression data
were normalized by center each array using the median over entire array as reference. A
probe were excluded if less than 20 % of expression data have at least a 1.5 -fold change
in either direction from probe's median value. The cut-off for significant differential
value between samples was 2.25 Log2 (ratio between geometric means of intensity for
each T-UCR probe). Statistical comparisons were performed using two-sample t-test,
Nominal significance level of each univariate test was 0.05. All data were submitted
using MIAMExpress to the ArrayExpress database.
Quantification and cellular localization of T-UCRs and miR-155 with real-time PCR
Each reverse transcriptase (RT) reaction contained 6 ng of purified and DNasetreated (turbo DNA-free, Ambion). Real-time PCR reactions included 2 μl of diluted RT
product (1:15 dilution). Reactions were incubated in an Applied Biosystems 7900HT
Fast Real-Time PCR system in 384-well plates. Total RNA was extracted from two
independent experiments and the Real-time PCR reactions for each T-UCR or miRNA
(10 μl) were performed in triplicate. The primers used are described in the Table S2. To
study the cellular localization of the T-UCRs, the qRT-PCR levels of Uc.160+,
Uc.283+A and Uc.346+ in the nucleus and cytoplasm were plotted as percentage,
defined as: percentage nucleus = (UCR quantified in RNA nuclear fractions)/(UCR
quantified in total fraction RNA) and percentage cytoplasm = (UCR quantified in RNA
cytoplasmic fractions)/(UCR quantified in total fraction RNA), using tRNAMet, U6 and
GADPH as loading controls of cytoplasm, nucleus and total cellular fractions,
respectively.
Quantitative chromatin immunoprecipitation (qChIP)
Quantitative PCR (qPCR) analysis was performed on an ABI 7900HT sequence
detection system (Applied Biosystems) with SYBR® Green. The amount of the
immunoprecipitated DNA sample was estimated as the average of results of three PCRs
and with reference to a standard curve generated from serially diluted sonicated
genomic DNA (INPUT). At least three biological replicates were performed and a
negative control (no antibody; Nab) was included for each qPCR experiment. Error bars
represent standard deviations. All qChIPs signals were normalized to the input (labeled
as IP/INPUT on the vertical axis), which should be considered arbitrary units on a
relative enrichment scale.
Chromatin accessibility using the Msp I assay
Growing cells were trypsinized and washed twice with cold phosphate buffer
saline (PBS). Cells were then resuspended in 1 ml of ice cold RSB, adding Nonidet P40
to 1% and kept on ice for 10 min. After incubation, cells were centrifuged for 5 min at
800g at 4°C. The supernatant was discarded and nuclei were then resuspended in RSB
plus NP40. Samples were centrifuged for 5 min at 800g at 4°C. Nuclei were washed
with RSB and centrifuged for 5 min at 800g at 4°C. After the supernatant was
discarded, the nuclei were finally resuspended in the corresponding buffer. For the MspI
accessibility assay, the nuclei were resuspended in 1x MspI buffer, so that there were
106 nuclei per 400 µl. Reactions were stopped by adding 100 µl of stop solution (20 mM
Tris-HCl pH 7.5, 0.6 M NaCl, 1% SDS, 10 mM EDTA and 400 µg/ml proteinase K)
and incubating at 37°C for 2 h. DNA was purified by phenol/chloroform extraction and
ethanol precipitation. Amplification and quantification of the amplicons was performed
by real-time PCR in an Applied Biosystems 7900HT Fast Real-Time PCR System,
using 40 ng of the DNA obtained for each amplification. The results shown are the
mean of at least two independent experiments amplifying each sample in triplicate in
each experiment.
Cell doubling time assay, trypan blue exclusion test of cell viability and cell cycle
analysis
Cell doubling time assay was performed as described (Melo et al., 2008).
Briefly, the same cell number was platted in 12 well plates by triplicate and left to grow
for 72 counting the cells every 8 hours. Cells were tripsinized and counted using a
Neubauer chamber at the time points upper mentioned. The doubling time was
calculated as follows: t (in hours) = h*ln(2)/ln(c2/c1). The trypan blue exclusion test is
used to determine the number of viable cells present in a cell suspension. Live cells
possess intact cell membranes that exclude trypan blue, whereas dead cells do not. The
mock and the Uc.283+A transfected cells were mixed with trypan blue and then visually
examined and counted. A mean of 230 cells was counted in three independent
experiments. For the cell cycle analysis, HCT-116 empty vector and Uc.283+A
transfected cells were trypsinized and fixed in 70% cold ethanol for 24 h at -20°C. Cells
were washed and resuspended in phosphate buffer saline solution containing 200 µg/ml
RNAse A and stained with 25 µg/ml propide iodide for 45 minutes at room temperature.
DNA content was analysed by flow cytometry (FACS Canto II; BD Biosciences,
Sparks, MD).
References
Melo SA, Ropero S, Moutinho C, Aaltonen LA, Yamamoto H, Calin GA et al. (2009).
A TARBP2 mutation in human cancer impairs microRNA processing and DICER1
function. Nat Genet 41:365-370.