Monday, July 23, 2012

Whole-transcriptome RNA-Seq analysis from archival FFPE tumor samples

Many FFPE tissue blocks containing samples related to  known disease states are archived in a multitude of hospitals worldwide. The RNA available from these blocks is not only limited in quantity but also generally severely degraded. In this report, Sinicropi et. al., discuss methods for RNA-Seq  using RNA isolated from archival FFPE samples with low input levels of RNA, and compare the results to data previously generated by RT-PCR analysis.

The authors used the MasterPure™ RNA Purification Kit to isolate total RNA from 136 FFPE primary breast cancer tumor specimens. The isolated RNA was used as input for the ScriptSeq™ mRNA-Seq Library Preparation Kit (now replaced by the ScriptSeq v2 Kit) for preparing whole-transcriptome libraries. Sequencing was performed on an Illumina HiSeq 2000 instrument. Analysis showed that the RNA-Seq data generated using 5- to 12-year-old FFPE tumor tissue compared very favorably with RT-PCR results published earlier, and enabled quantification of transcripts with accuracy and sensitivity sufficient for biomarker discovery. The study identified >1,300 transcripts associated with breast cancer risk; more than half of the RNAs identified as prognostic were noncoding. Thus, the study demonstrated the value of a whole-transcriptome approach, and the results obtained should prove valuable in future screens of biomarkers associated with breast cancer recurrence risk.

ResearchBlogging.orgSinicropi D et al. (2012). Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue. PloS one, 7 (7) PMID: 22808097

Monday, July 16, 2012

Retroviral gene expression in multiple sclerosis

Multiple sclerosis (MS) is a devastating illness with no known cause or cure. Recently, Laska et al. revealed that onset of the illness appears to increase expression of a specific retrovirus, HERV-Fc1, that belongs to the HERV-H/F family.

The study included investigating expression of certain components of HERV-Fc1 by qRT-PCR. During the studies, standards for various expressed transcripts of the virus were generated using the Durascribe® T7 Transcription Kit. The Durascribe Kit synthesizes RNA that contains 2'-fluorouracil and 2'-fluorocytidine, rendering transcripts resistant to degradation by ribonucleases A, B, and C. The standards were used as controls to determine the extracellular HERV-Fc1 RNA load in normal and MS individuals. The analysis showed that the expression of a capsid (gag) protein of HERV-H/F was significantly increased in CD4+ (P <0.001) and CD8+ (P <0.001) T lymphocytes, and in monocytes (P = 0.0356) from MS patients with active disease.

The importance of T cells in MS induction and further progression has been well documented. The methods developed in this study will enable further investigation of differential expression of endogenous retroviral markers in HERV-Fc1 biology related to MS, and possibly other autoimmune disorders.

ResearchBlogging.orgLaska MJ et al (2012). Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis. Journal of virology, 86 (7), 3713-22 PMID: 22278236

Thursday, July 12, 2012

New products: ScriptSeq Complete Kits

Epicentre’s ScriptSeq™ Complete Kits are now available. The kits combine Epicentre's industry-leading Ribo-Zero™ technology and rapid ScriptSeq v2 method for seamless, end-to-end preparation of directional RNA-Seq libraries in 1 day. The ScriptSeq Complete Kits use 1-5 μg of total RNA, and the ScriptSeq Complete Kits—Low Input use 100 ng to 1 μg of total RNA. The Low Input kits are especially suitable for RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue samples.