Supplementary Information
Methods for RNA and DNA extraction
10mg of tissue was homogenized using a rotor-strator homogenizer in 1mL Trizol. Chloroform was
added according to the manufacturers protocol, and the phases separated by centrifugation. The
upper aqueous phase was removed and total-RNA purified from it using a Micro-to-Midi RNA
purification kit (Invitrogen) according to the manufacturers protocol.
DNA was purified from 10mg of tissue using a Micro-to-Midi DNA Purification Kit (Qiagen),
following homogenisation in phosphate buffered saline. This was performed according to the
manufacturer’s protocol aside from an extension of proteinase-K digestion to 1 hour. Samples were
assessed for purity and concentration by spectrophotometry.
FOXP1 Immunohistochemistry
Scoring of the FOXP1 immunostaining was performed as previously described by Banham et al.
(2005). Samples were scored by two independent assessors as follows: failed, complete lack of
nuclear staining of internal control small lymphocytes; 0, <10% staining of large tumoral B-cells; 1,
10% to 30% of the large B cells with nuclear staining; 2, 31% to 50% nuclear staining of large B
cells; and 3, >50% nuclear staining of large neoplastic B cells. Two cases showed an unusual
cytoplasmic staining pattern and were not included in this analysis. For the purposes of this study,
scores of 0 and 1 (<30% of the cells positive) were considered negative and scores of 2 and 3
(>30% of the cells staining) were considered positive for FOXP1 expression. Duplicate cores that
did not have identical scores for FOXP1 staining were averaged.
Detailed Methods for qPCR
The most stable reference gene for FL samples was determined as previously described by
Vandesompele et al. (2002). This method utilised intron-spanning primers in order to enable the
detection of genomic DNA contamination by melt-curve analysis. cDNA was reverse-transcribed in
triplicate from 1µg of total-RNA using the SuperScript III™ Reverse Transcription System
(Invitrogen) and random hexamer primers according to the manufacturers protocol, and the
reactions pooled. qPCR was performed on a RotorGene 6000 (Corbett) using reactions consisting of
1X iQ-SYBR Green supermix (BioRad), 10μM of each primer (Supplementary Table 1) and 1μL of
cDNA. Melt-curve analysis was performed for each reaction in order to assure amplification
specificity and the lack of primer-dimer formation. CT values were averaged for triplicate reactions
and used to calculate ΔCT values for each sample. Isoform 2 expression fold-changes between case
and control samples were calculated using the 2-ΔΔCT method [Livak and Schmittgen, 2001].
Assays were designed to be specific for exons 1 (N-terminal), 6a (Isoform-2) and 20 (C-terminal) of
the FOXP1 gene. Movement of the C-terminal assay qPCR threshold cycle (CT) is thus only
influenced by the abundance of both full-length and truncated isoforms, whereas the N-terminal
assay CT is influenced only by the abundance of full-length isoforms. Changes in the abundance of
full-length isoforms would therefore alter CT values of both the N-terminal and C-terminal assays
and cause no change to the resulting FOXP1ΔCT. In contrast, changes in the abundance of truncated
isoforms would alter only the C-terminal assay CT, resulting in an increased ΔCT value with
increasing relative abundance of truncated isoforms and decreasing ΔCT with decreasing relative
abundance of truncated isoforms.
FOXP1ΔCT = N-terminal CT – C-terminal CT
C-terminal ΔCT = C-terminal CT – RPL13A CT
Isoform-2 ΔCT = Exon 6a CT – RPL13A CT
Detailed Methods for Illumina Microarrays:
Human total RNA was quality ascertained with an Agilent Bioanalyser 2100 using the NanoChip
protocol. A total of 500ng was labelled using the Ambion Total Prep RNA amplification kit (Cat.
No. – IL1791). The quantity of labelled product was ascertained with Agilent Bioanalyser 2100
using the NanoChip protocol. A total of 1.5ug of labelled cRNA was then prepared for
hybridisation to the Sentrix Human-6 Expression Beadchip (v2.0) by preparing a probe cocktail
(cRNA @ 0.05ug/ul) that includes GEX-HYB Hybridisation Buffer.
A total hybridisation volume of 30ul is prepared for each sample and 30ul loaded into a single array
on the Sentrix Human-6 Expression Beadchip (v2.0). A total of 6 different labelled samples can be
loaded into 6 individual arrays per beadchip. The chip is hybridised at 58C for 16 hours in an oven
with a rocking platform. After hybridisation, the chip is washed using the appropriate protocols as
outlined in the illumina manual. Upon completion of the washing, the chips are then coupled with
Cy3 and scanned in the illumina BeadArray Reader. The scanner operating software, BeadStudio,
converts the signal on the array into a TXT file for analysis.
NFκB-Associated Genes
A literature review was conducted in order to define NFκB-associated genes [Bauerle and
Baichwal, 1997; Darnay et al., 1998; Ghosh et al., 1998; Israel, 2000; Pomeerantz and Baltimore,
1999; Shaffer et al., 2001; Shibuya et al., 1996; Verma et al., 1995]. As a result, 137 genes were
associated with NFκB signalling and included in our analyses (Supplementary Table 3).
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