Expression profiling with RNA from formalin-fixed, paraffin-embedded material

<p>Abstract</p> <p>Background</p> <p>Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.</p> <p>Results</p> <p>We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.</p> <p>Conclusion</p> <p>We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.</p

Cellular senescence of white blood cells in very long-term survivors after allogeneic hematopoietic stem cell transplantation: the role of chronic graft-versus-host disease and female donor sex

In this single-center, cross-sectional study, we evaluated 44 very long-term survivors with a median follow-up of 17.5 years (range, 11-26 years) after hematopoietic stem cell transplantation. We assessed the telomere length difference in human leukocyte antigen-identical donor and recipient sibling pairs and searched for its relationship with clinical factors. The telomere length (in kb, mean +/- SD) was significantly shorter in all recipient blood cells compared with their donors' blood cells (P > .01): granulocytes (6.5 +/- 0.9 vs 7.1 +/- 0.9), naive/memory T cells (5.7 +/- 1.2 vs 6.6 +/- 1.2; 5.2 +/- 1.0 vs 5.7 +/- 0.9), B cells (7.1 +/- 1.1 vs 7.8 +/- 1.1), and natural killer/natural killer T cells (4.8 +/- 1.0 vs 5.6 +/- 1.3). Chronic graft-versus-host disease (P > .04) and a female donor (P > .04) were associated with a greater difference in telomere length between donor and recipient. Critically short telomeres have been described in degenerative diseases and secondary malignancies. If this hypothesis can be confirmed, identification of recipients at risk for cellular senescence could become part of monitoring long-term survivors after hematopoietic stem cell transplantation

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-7

gene-specific primers (hatched boxes). Gene expression was measured from an equivalent of 4 ng of RNA by QPCR for five reference genes (GAPDH, GUSB, RPLP0, TFRC and UBB). Pearson correlations were computed between matched Cts for the five reference genes and each tumor RNA isolated from FF (A) and FFPE material. Shown are correlations between intact RNA and RNA isolated from FFPE material according to the RNeasy FFPE protocol (A versus B), intact RNA and RNA isolated from FFPE material according to the ncLysis system (A versus C) and intact RNA and RNA isolated according to our own protocol (A versus D).<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-4

RC, RPL7A, RPS11, RPS23 and UBB). Results based on intact RNA derived of FF material (A) and based on RNA isolated according to our own protocol from FFPE material (B) are depicted for all the 14 tumors. The Ct values for 4 ER-related genes (BCL2, CEPG1, ESR1 and PGR) are shown for comparison (left).<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-5

Lts of intact RNA (○) and FFPE-derived RNA (△, own protocol) as described in the Methods section. They are shown separately for each of the 14 tumors.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-0

gene-specific primers (hatched boxes). Gene expression was measured from an equivalent of 4 ng of RNA by QPCR for five reference genes (GAPDH, GUSB, RPLP0, TFRC and UBB). Pearson correlations were computed between matched Cts for the five reference genes and each tumor RNA isolated from FF (A) and FFPE material. Shown are correlations between intact RNA and RNA isolated from FFPE material according to the RNeasy FFPE protocol (A versus B), intact RNA and RNA isolated from FFPE material according to the ncLysis system (A versus C) and intact RNA and RNA isolated according to our own protocol (A versus D).<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-6

Reast cancers and visualized in a scatter plot. The ER score was determined from four genes, the HER2 score from 2 and the proliferation score from 5 genes (see Methods). Tumors are positioned according to their ER score (x-axis) and HER2 score (y-axis). Proliferation scores are color coded. The histological ER status is indicated by a "-" or "+" sign next to the tumor numbers in the plot. The results were computed from intact RNA derived of FF material (A) and RNA isolated from FFPE material according to our own protocol (B). Individual scores for each tumor are given in Table 2.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-3

3 and BM36 (panel A) are two separate RNAs isolated from tissue block "BM", D33 and D36 are RNAs isolated from block "D" (panel B). For comparison, 45T and 56T originate from two distinct tumors isolated from one patient (panel C). Gene expression was measured by QPCR for 24 genes and raw Ct values are shown for each gene measured from the two matching RNAs.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p

Expression profiling with RNA from formalin-fixed, paraffin-embedded material-2

RC, UBB) for intact RNA (◇, FF) and for RNA isolated from matched FFPE material according to the protocols of Qiagen (□, Q), Applied Biosystems (△, AB) and our own (○, own). Individual mean Cts of the 14 tumors and summarized box plots of Cts are shown in panel A and panel B, respectively. Tumors are aligned according to increasing Ct in FFPE-derived RNA (Qiagen protocol).<p><b>Copyright information:</b></p><p>Taken from "Expression profiling with RNA from formalin-fixed, paraffin-embedded material"</p><p>http://www.biomedcentral.com/1755-8794/1/9</p><p>BMC Medical Genomics 2008;1():9-9.</p><p>Published online 19 Apr 2008</p><p>PMCID:PMC2359756.</p><p></p