Mutation detection by real-time PCR: a simple, robust and highly selective method.

BACKGROUND: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. METHODOLOGY/PRINCIPAL FINDINGS: The method we describe is based on the widely used TaqMan real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. CONCLUSIONS/SIGNIFICANCE: ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability

Data from: Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue

We report a method for Selective Depletion of abundant RNA (SDRNA) species from Human total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, here demonstrating removal of ribosomal and mitochondrial transcripts from clinical FFPE tissue RNA archived up to 20 years. Importantly, SDRNA removes 98% of targeted RNAs while preserving relative abundance profiles of non-targeted RNAs, enabling routine whole transcriptome analysis of clinically valuable archival tissue specimens by Next-Generation Sequencing

Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue

<div><p>We report a method for <u>S</u>elective <u>D</u>epletion of abundant <u>RNA</u> (<u>SDRNA</u>) species from Human total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, here demonstrating removal of ribosomal and mitochondrial transcripts from clinical FFPE tissue RNA archived up to 20 years. Importantly, SDRNA removes 98% of targeted RNAs while preserving relative abundance profiles of non-targeted RNAs, enabling routine whole transcriptome analysis of clinically valuable archival tissue specimens by Next-Generation Sequencing.</p> </div

gene_counts

Gene level read counts for the eight libraries reported in the manuscript

Scatterplots of RefSeq transcript abundance: Depleted vs. Undepleted.

<p>Log₁₀ read counts are plotted. Transcripts with zero reads are excluded from analysis. An identity line is shown in each plot for reference. Sub-populations are indicated as follows: Black cross – sno-, sc-, sn- and mir-RNAs from RefSeq; Black and red circles – Genes homologous to SDRNA and SDRNA2 probes, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042882#pone.0042882.s007" target="_blank">Table S4</a>); Light blue circles – All other RefSeq transcripts. FF – Fresh-frozen; FFPE – Formalin-fixed, paraffin-embedded. (<b>a</b>) FF Breast RNA: not depleted vs polyA+ (<b>b</b>) FF Breast RNA: not depleted vs SDRNA1 and (<b>c</b>) FF Breast RNA: not depleted vs SDRNA2. (<b>d</b>) FFPE RNA pool: not depleted vs SDRNA1 and (<b>e</b>) FFPE RNA pool: not depleted vs SDRNA2.</p

Read density of targeted regions.

<p>Read density plotted across regions targeted for depletion and compared between different depletion methods. Regions targeted for depletion (SDRNA1 = green, SDRNA2 = red and green) are indicated at the bottom of each figure. Density values were computed using igvtools count (<a href="http://www.broadinstitute.org/igv/igvtools" target="_blank">www.broadinstitute.org/igv/igvtools</a>) and plotted using UCSC’s Genome Browser. FF – Fresh-frozen; FFPE – Formalin-fixed, paraffin-embedded.(a) Reads are mapped to chrUn_gl000220, an unplaced genomic contig from hg19 containing the 13 kb rRNA transcript described in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042882#pone-0042882-g001" target="_blank">Figure 1a</a>. (b) Reads are mapped to the human mitochondrion from hg18 (Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042882#pone-0042882-g001" target="_blank">Figure 1b</a>).</p

Proportion of reads uniquely-mapping to rRNA or non-rRNA categories.

<p>The rRNA category represents all rRNAs encoded in both nuclear and mitochondrial genomes (see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042882#pone-0042882-g001" target="_blank">Figure 1</a>). Non-rRNA includes all other uniquely-mapping reads.</p

Distribution of uniquely-mapped reads across broad RNA classes. rRNA class includes all reads mapping to the 13 kb transcript shown in Supplementary

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042882#pone-0042882-g001" target="_blank"><b>Figure 1a</b></a><b>.</b> Mito class refers to all reads mapping to the mitochondrial genome shown in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042882#pone-0042882-g001" target="_blank">Figure 1b</a>. The Intergenic class encompasses all remaining reads not mapping to RefSeq annotations or their associated introns. FF – Fresh-frozen; FFPE – Formalin-fixed, paraffin-embedded. (a) polyA+ FF breast RNA.(b) SDRNA2 FF breast RNA. (c) SDRNA2 FFPE RNA pool.</p