Tumor and normal tissue samples
Frozen HNSCCs and the corresponding adjacent normal tissues were obtained from five patients treated at MD Anderson Cancer Center as part of a randomized trial that included surgical resection of the primary tumor and postoperative radiotherapy (13
). The patients had histologically proven locally advanced squamous cell carcinoma of the oral cavity or laryngopharynx and were classified as having a high-risk for recurrence based upon surgical and pathologic criteria (see ) and for whom no subsequent recurrence was seen. Tumor and normal tissue were obtain as follows: 1)Grossly normal mucosa from the farthest resection margin was carefully excised and subjected to frozen section evalution to exclude dysplasia and the presence of inflammatory cells. The surface squamous epithelium of an adjacent area was then carfully peeled off the submucosa and placed immediately in liquid nitrogen. This process insured more than 90% epithelial cells in each “normal” sample. 2)Tumor tissue was also harvested from the periphery of the tumor mass and a thin slice was used for frozen section evaluation to assess the quality and quantity of the specimen. All of the samples used contained an excess of 80% viable tumor cells.
Approximately 100mg of frozen tissue was lysed in approximately 2ml of Trizol (Invitrogen, Carlsbad, CA) using a motorized Pro200 homogenizer (Pro Scientific, Oxford, CT). Extreme care was taken to avoid any partial thawing of the frozen sample prior to lysis. The Trizol lysate was subjected to organic extraction as described in the manufacturer's protocol. The aqueous phase that contained the total RNA was carefully removed and used to enrich for small RNAs (< 200 nt). The enrichment was accomplished by first immobilizing large RNAs on a glass fiber filter (Ambion, Austin, TX), then eluting with a relatively low ethanol concentration and collecting the flow-through that contained mostly small RNA species. More ethanol was added to this flow-through, and the mixture was passed through the second glass filter where the small RNAs were immobilized. The second filter was then washed several times and the small RNA enriched sample eluted with a low ionic strength solution. Large RNAs (>200 nt) from the first filter were also eluted after a few washes and used to check the integrity of the RNA source by running an aliquot on an Agilent 2100 Bioanalyzer automated electrophoresis platform (Agilent Technologies, Santa Clara CA). (Total RNA collected for gene expression analysis was also examined on this platform.) Approximately 100μg of the small RNA enriched samples were fractionated by passing through a Flash PAGE fractionator (Ambion, Austin, TX). The flash PAGE™ Fractionators can fractionate mature miRNAs from other small RNA species in just 12 minutes. This system has been designed and optimized to separate mature miRNA (19–23 nt) away from longer precursor molecules and provides an efficient alternative to standard, time-consuming polyacrylamide gel electrophoresis and subsequent gel elution. The fraction containing the miRNAs (<40nt) was recovered and cleaned using Ambion reagents following the manufacturer's protocol. miRNA samples were quantified by placing 1μl of the samples in the ND-1000 Nanodrop (NanodropTech Wilmington, DE).
miRNA labeling and hybridization
To determine the amount of enriched miRNA needed to produce sufficient fluorescence signal intensity, preliminary hybridizations were performed using 1, 2, 5, and 10μl of enriched miRNA (68ng/μl). From these results, 600ng was chosen as the amount of enriched miRNA to be used for subsequent hybridizations. The mirVana miRNA labeling system (Applied Biosystems, Foster City, CA) was used to add a 3′ amine-modified tail to each miRNA target sample that was then labeled with NHS-esters of Cy5 fluorescent dye (GE Healthcare Piscataway, NJ) according to the manufacturer's protocol.
The mirVana miRNA bioarray system from Ambion (Austin, TX) was used to determine miRNA expression. The preprinted mirVana miRNA bioarray V1 has probes targeting a selection of human (213), mouse (51), and rat (30) miRNAs from the miRBase Version 8.0 Sanger miRNA Registry, as well as Ambi-miRs (105) that are exclusive to Ambion. Twenty-six controls are also present on each array. Each target sample was applied to two arrays and the arrays were sealed in hybridization chambers (Corning Acton, MA). The arrays were incubated overnight at 42°C in a rocking hybridization oven. The arrays were then washed in a low stringency wash buffer twice for 30s, followed by a high stringency wash buffer twice for 30s, then quickly rinsed twice in water and dried by centrifugation.
The dried arrays were scanned using the ArrayWorx Autoe scanner (Applied Precision, Issaquah, WA) and the fluorescence quantified using ArrayVision software (Imaging Research, St. Catherines, Ontario, Canada). The quantified data were processed using the statistical package Splus 6 (Insightful, Seattle, WA). Local estimates of the background signal were subtracted from raw signal intensities for each array feature. Based on a histogram of the maximum spot intensity and maximum S/N, the features were filtered for a maximum S/N>4 as being trusted features. Of these, 249 features were included as trusted and the median intensities of these features were used to normalize the data on each array. The normalized data was logarithm transformed to base two and the mean data of the replicates was determined. The log ratio values were calculated for the tumor and corresponding paired normal samples. Differentially regulated miRNA between the two samples were identified using a paired t test.
Quantitative RT-PCR of miRNA
Quantitative RT-PCR was performed using the TaqMan MiRNA assay system (Applied Biosystems). The miRNA enriched fraction was used instead of the total RNA so as to include the normalization control miRNA Rnu-44, which is longer than the regular miRNAs. Briefly, about 2 ng of the miRNA enriched fraction (< 200 nt) was subjected to a RT reaction using a miRNA-specific looped primer according to the manufacturer's protocol in order to make cDNA. Subsequent PCR used miRNA specific forward and reverse primers along with an appropriate quantity of RT cDNA product and Taqman universal mix. The PCR reaction for each miRNA-cDNA, was run in quadruplicate. A negative control without template was included in parallel to assess the specificity of the PCR reaction. PCR was carried out in AB7900 (Applied Biosystems) in a 20μl volume with the following thermal cycling parameters: enzyme activation step at 95°C for 10 min; 40 cycles of denaturation at 95°C for 15sec; and annealing/extension at 60°C for 60 sec. All other conditions used the manufacturer's values.
Data acquired from the PCR reactions was analyzed using SDS2.3 software (Applied Biosystems), which is based on the comparative CT (threshold cycle) – ΔΔCT method. Rnu-44 normalized ΔΔCT values obtained for each miRNA from the normal samples was compared with the corresponding miRNA from the corresponding tumor sample. Comparisons are described as log values of the ratio of miRNA expression in tumor samples vs adjacent normal tissue.
mRNA gene expression profiling
mRNA hybridization of the tumor and normal tissues were performed as described in (14
) using the spotted Oligonucleotide microarray chips produced by the Wiegand Radiation Oncology Core at MDACC. These arrays contain approximately 18,000 human oligonucleotides (SigmaGenosys, St.Louis, MO). A 22.5μg aliquot of the >200nt RNA fraction from either the tumor or normal tissue was compared against a reference Human Universal RNA (Stratagene Biocrest La Jolla, CA). All samples were labeled using the SuperScript indirect labeling kit (Invitrogen Corporation Carlsbad, CA) to produce cDNA for each slide. The cDNA was labeled with either a Cy3 (tumor or normal tissue) or Cy5 (Universal Reference) monofunctional reactive dyes (GE Healthcare Piscataway, NJ). Labeled Cy-cDNA was then mixed 1:1 with HybIt Hybridization buffer (Telechem Inc, Sunnyvale, CA). The target samples were injected onto the oligo array chips that had been loaded into a Lucidea SlidePro automated hybridization station (GE Healthcare, Piscataway, NJ). The arrays were incubated for 16 h at 42°C. The arrays were first washed at 50°C first with 4ml of 1 X SSC and 0.2% Sarkosyl, then with 2ml of 0.1 X SSC and 0.2% Sarkosyl, followed by a final Wash at room temperature with 2 ml of 0.1 X SSC. The arrays were then rinsed with isopropanol and air dried before being scanned with the Array Worx scanner.
The initial data analysis was carried out as described in (14
). A simple two-sample t-test was used to score the differential gene expression between tumor samples and the corresponding normal tissues using a beta-uniform mixture model (15
) and false discovery rate for feature cutoff.