Appendix S1 - Supplementary methods, data and references
The microarray technology that identified Rif as potentially significant in transformed
lymphoma has inherent limitations regarding precision, scale, and signal-to-noise ratio,
We therefore chose to confirm and extend the results of our findings by directly
measuring RIF mRNA expression in a range of lymphoid tissue.
Isolation of lymphocyte subsets
Enriched lymphocyte subsets were derived from tonsils and peripheral blood as
previously described (Klein, et al 2003). Tonsils were obtained with Institutional Review
Board (IRB) approval (IRB# 107233) from routine tonsillectomies performed at the
Primary Children's Medical Center (Salt Lake City, UT) as fully anonymized residual
material. Specimens were kept on ice following surgical removal and minced, with the
mononuclear population isolated by Ficoll-Isopaque density centrifugation. Three B-cell
subpopulations were isolated from tonsils by magnetic cell separation by using the
MidiMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, naïve Bcells were isolated by depletion of GC B-cells (CD10, CD27), memory B-cells (CD27),
plasma cells/blasts (CD27), and T-cells (CD3), followed by a positive enrichment of IgDpositive cells. Memory B-cells were purified by depletion of GC B-cells (CD10, CD38),
plasma cells/blasts (CD38), and T-cells (CD3), followed by a positive enrichment of
CD27-positive cells. Germinal center B-cells were isolated in a single step by
magnetically labeling CD10-positive cells. Purified T-cells were derived from peripheral
blood obtained from healthy volunteers, with IRB approval. T-cells were isolated in a
single step by magnetically labeling CD3-positive cells.
Cell culture
Cell lines obtained from ATCC (Manassas, VA) and DSMZ (Braunschweig, Germany)
were cultured under standard conditions as previously described (Fillmore, et al 2002,
Robetorye, et al 2002). We studied three t(14;18)-positive cell lines (SUDHL-4, SUDHL6 and Karpas 422), six t(14;18)-negative B-cell NHL cell lines (SUDHL-5, SUDHL-7,
SUDHL-8, SUDHL-9, OCI-LY8 and OCI-LY10), one t(8;14)-positive B-cell NHL cell
line (Raji), two t(2;5)-positive T-cell ALCL cell lines (SUDHL-1, Karpas 299), and one
T-cell ALL cell line (Jurkat).
Tissue samples
Lymphoma patient samples were obtained from the Sunnybrook and Women's College
Health Sciences Centre and the Cross Cancer Institute as previously described (ElenitobaJohnson, et al 2003). Thirty-seven primary lymphoma samples derived from patients with
FL (n=5), de novo DLBCL (n=5), FL to DLBCL transformed lymphoma (FTL) (n=5),
chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n=4),
anaplastic large cell lymphoma (ALCL) (n=8), mantle cell lymphoma (MCL) (n=5), and
T-cell acute lymphoblastic leukemia (T-ALL) (n=5). Benign tissue from reactive lymph
nodes (n=2) and tonsillar tissue (n=3) were obtained as anonymized residual tissue from
histologically confirmed non-malignant lymph node biopsies done at the University of
Utah Medical Center, and routine tonsillectomies performed at the Primary Children's
Medical Center (Salt Lake City, UT). All samples were snap frozen and stored at –70o C
until RNA extraction, or extraction was done from fresh tissue stored on ice. Tissues
were obtained and studied with IRB approval (IRB # 107233).
RNA extraction
cDNA template was prepared from four enriched populations of lymphoid cells isolated
by immunomagnetic bead selection, thirteen lymphoma/leukemia cell lines, five
nonmalignant lymphoid tissues (reactive lymph nodes and tonsillar tissue) and thirty-six
malignant pathological tissue samples. Total RNA was extracted using Trizol™
(InVitrogen, Carlsbad, CA) according to manufacturer's instructions. First-strand cDNA
was synthesized using random hexamers and the SuperScript II reverse transcriptase
(InVitrogen, Carlsbad, CA) according to manufacturer's instructions. mRNA and cDNA
was quantitated spectrophotometrcally and subjected to electrophoretic quantitation and
analysis to ensure quality and yield.
Quantitative RT-PCR
PCR primer sequences for RIF and control genes were either derived from previous
studies or designed from mRNA FASTA sequence analyzed by the primer3 program
(Rozen and Skaletsky 2000). Primers were designed for products in the 150-200bp range,
preferentially from sequence closer to the 5' end of each gene, and with forward and
reverse primers on different exons. (Table SI). PCR conditions were checked and
optimized using a range of magnesium concentrations and annealing temperatures.
Real time quantitative RT-PCR was carried out in 96-well plates using an ABI PRISM
7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly,
cDNA samples were diluted to 10ng/l cDNA in nuclease-free water. Amplification
mixtures totaling 20 l per reaction contained 75ng template, DyNAmo HS 2X SYBR
Green master mix buffer (Finnzymes Oy, Espoo Finland), 1x ROX passive reference dye,
and 500nM forward and reverse primer. Cycling conditions entailed 15 min polymerase
activation at 95oC followed by 40 cycles at 94oC for 15 sec, 55oC for 20 sec and 72oC for
20 sec, with quantification detection at the 72oC extension step. A final quantified
denaturing step from 55oC to 95oC produced a melting curve to determine product
specificity. All samples were run in triplicate, with a no-template negative control.
Threshold cycle (Ct) was determined as the fractional cycle number at which the amount
of amplified target reached a fixed fluorescence threshold. Sequence Detection Software
(v 2.1) (Applied Biosystems, Foster City, CA) results were exported as tab-delimited text
into Excel (Microsoft, Redmond, WA) for further analysis.
PCR efficiency of each primer set was determined by QRT-PCR as above using five 8fold serial dilutions of SUDHL-6 cDNA template (375ng, 46.88ng, 5.86ng, 0.73ng,
0.09ng) and a no-template negative control using the above conditions.
Statistical analysis
PCR efficiency of each primer set was derived from the slope of serial dilutions with
efficiency (E) calculated according to the equation: E = 10[–1/slope] (Rasmussen 2001).
Inter-assay variability was assessed by standard variance calculations. These calculations
as well as the relative expression ratio calculated from RT-PCR efficiency using the Ct of
RIF samples are derived from a previously described equation (Pfaffl 2001), using the
equation:
(Etarget)Cttarget
ratio = ------------------------------(Ereference)Ctreference
So long as E values remain comparable, with PCR efficiency approaching 2n copies per
cycle, the ratio (R) can be simplified to:
R = 2Ct (target-reference)
Although most studies utilize a single "housekeeping" gene as reference, several studies
have questioned the assumption that a single gene will maintain constant expression
levels among samples and tissue types (Suzuki, et al 2000, Thellin, et al 1999,
Vandesompele, et al 2002). Therefore, rather than use a single control Ct reference to adjust
for total mRNA input, we used the geometric mean Ct of three housekeeping genes for
increased stringency in normalization. Using the geometric mean Ct of CYCA, UBC and
TBP to get a Ct reference value, we normalized for input mRNA to standardize reactions in
respect to RNA integrity, variations in the reverse transcriptase and polymerase
amplification reactions, and sample loading.
ANOVA was derived and rendered using StatView 4.5.1 for Apple Macintosh OS 9
(SAS, Cary NC). Data was evaluated statistically by analysis of variance to determine
relative quantification between samples. Calculations used for pairwise variation of
control genes CYCA, TBP and UBC, and internal control gene-stability were also derived
from equations as described (Vandesompele, et al 2002). A spreadsheet of the raw data
is available on request in Microsoft Excel format.
Supplementary Data
All PCR assays were robust, with efficiencies above 95%. Results were highly
reproducible; for all runs done in triplicate, maximum inter-assay variation (IAV) was
less than 5% with mean and median IAV less than 1%. Pairwise variation and internal
control gene-stability calculations for showed low inter-control variation, confirming
their suitability for normalization. Based on serial dilution analysis, efficiency E of
primers was determined to be ~2 for PCR amplifications with all primer sets.
RIF quantification and ANOVA for lymphoma/leukemia cell lines and enriched B-cell
populations are shown in Table SII and Fig S1. Results for primary lymphoma and
benign lymphoid tissue samples are shown in Table SIII and Fig S1.
Supplementary References
Elenitoba-Johnson, K.S., Jenson, S.D., Abbott, R.T., Palais, R.A., Bohling, S.D., Lin, Z.,
Tripp, S., Shami, P.J., Wang, L.Y., Coupland, R.W., Buckstein, R., PerezOrdonez, B., Perkins, S.L., Dube, I.D. & Lim, M.S. (2003) Involvement of
multiple signaling pathways in follicular lymphoma transformation: p38-mitogenactivated protein kinase as a target for therapy. Proceedings of the National
Academy of Sciences of the United States of America, 100, 7259-7264.
Fillmore, G.C., Lin, Z., Bohling, S.D., Robetorye, R.S., Kim, C.H., Jenson, S.D.,
Elenitoba-Johnson, K.S. & Lim, M.S. (2002) Gene expression profiling of cell
lines derived from T-cell malignancies. FEBS Letters, 522, 183-188.
Klein, U., Tu, Y., Stolovitzky, G.A., Keller, J.L., Haddad, J., Jr., Miljkovic, V., Cattoretti,
G., Califano, A. & Dalla-Favera, R. (2003) Transcriptional analysis of the B cell
germinal center reaction. Proceedings of the National Academy of Sciences of the
United States of America, 100, 2639-2644.
Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time
RT-PCR. Nucleic Acids Research, 29, e45.
Rasmussen, R. (2001) Rapid Cycle Real-time PCR, Methods and Applications. Springer
Press, Heidelberg.
Robetorye, R.S., Bohling, S.D., Morgan, J.W., Fillmore, G.C., Lim, M.S. & ElenitobaJohnson, K.S. (2002) Microarray analysis of B-cell lymphoma cell lines with the
t(14;18). Journal of Molecular Diagnostics, 4, 123-136.
Rozen, S. & Skaletsky, H. (2000) Primer3 on the WWW for general users and for
biologist programmers. Methods in Molecular Biology, 132, 365-386.
Suzuki, T., Higgins, P.J. & Crawford, D.R. (2000) Control selection for RNA
quantitation. Biotechniques, 29, 332-337.
Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T.,
Igout, A. & Heinen, E. (1999) Housekeeping genes as internal standards: use and
limits. Journal of Biotechnology, 75, 291-295.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. &
Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR
data by geometric averaging of multiple internal control genes. Genome Biology,
3, Research0034.0031-0034.0011.
Supplementary Figure Legend
Figure S1: ANOVA analysis interaction bar plot for mean RIF expression of isolated homogeneous cell
populations (purified cells and cell lines) showing the effect of a: origin and b: lineage. Purified =
immunomagnetic bead separated B- and T-cell populations; cell line = lymphoma/leukemia cell lines,
described in supplementary Methods. Mean RIF expression of primary tissue samples showing the effect
of c: diagnosis and d: lineage. Abbreviations are described in Table III. Bars show  1 standard error.
Supplementary Table Legends
Table SI: Genes and primers used in this study. Please refer to supplementary Methods for description.
Table SII: Relative expression of RIF in purified B- and T-cells and cell lines derived from B- and T-cell
malignancies. RIF values are normalized to the geometric mean (Gm) of three control genes (cyclophilin
A, TATA-binding protein and ubiquitin C) previously demonstrated to have minimal variability among
lymphoid, leukocyte and bone marrow tissue, and represent relative values. B-NHL = B-cell non-Hodgkin
lymphoma; ALCL = anaplastic large cell lymphoma; T-ALL = T-cell acute lymphoblastic leukemia.
Characteristic chromosomal translocations found in the cell of origin are described, when available.
Table SIII Relative expression of RIF in malignant and non-malignant lymphomatous tissue samples. RIF
values are normalized to the geometric mean (Gm) of three control genes as described in the text, and
represent relative values.