Thursday, February 23, 2012

An improved RNA-Seq library prep workflow for FFPE RNA

Preparing high-quality, rRNA-depleted RNA-Seq libraries using RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is notoriously difficult. FFPE RNA is typically degraded (resulting in small fragment sizes) and is not compatible with poly(A)+ enrichment methods to remove contaminating rRNA. We have recently evaluated Ribo-Zero™ Kits and the ScriptSeq™ v2 Kit for the preparation of RNA-Seq libraries from FFPE RNA.

In this study, two different FFPE RNA samples were treated with either the Ribo-Zero Kit (Human/Mouse/Rat), which removes cytoplasmic rRNA, or the Ribo-Zero Gold Kit (Human/Mouse/Rat), which removes both cytoplasmic and mitochondrial rRNA. The rRNA-depleted RNA was then used to prepare RNA-Seq libraries using the ScriptSeq v2 Kit. Libraries were sequenced on an Illumina® GAIIx sequencer. 

Figure 1 (click to enlarge). Summary of sequencing metrics from FFPE RNA libraries.

As shown in Figure 1, the workflow yields high-quality RNA-Seq libraries, with excellent removal of rRNA and >98% directionality from FFPE RNA. Quality metrics surpass those obtained from FFPE RNA using standard ligation-based library preparation workflows.

Tuesday, February 14, 2012

Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

A recently published report by Murakami et al. (Nucleic Acid Res. doi:10.1093/nar/gkr909) describes a novel RNA sequence-specific detection method that uses a Phi 29-based rolling-circle amplification system, with potential utility in molecular diagnostic applications. The method involves using a sequence-specific circular probe system that is synthesized using CircLigase™ ssDNA Ligase. The original method, called primer generation-rolling circle amplification (PG-RCA), was developed to detect low amounts of DNA (see Epicentre Forum 16-2, 373K PDF).

The current method combines PG-RCA with the use of three-way junction (3WJ) probes designed to hybridize to short complementary sequences on the target RNA. The probes do not interact in the absence of target RNA. Next, the addition of DNA polymerase and a "nickase" enzyme results in an isothermal cycling process to generate signal primers. These signal primers are used in the final step of PG-RCA to amplify the desired target.

The level of amplification was determined by SYBR Green dye binding. Fluorescence intensity increases as more double-stranded DNA is accumulated. A negative result (low to no fluorescence) is obtained when the circular probe is not able to hybridize to the target sequence on the RNA. The authors were able to detect 15.9 zmol (9.55 × 103 molecules) of synthetic RNA or 143 zmol (8.6 × 104 molecules) of in vitro transcribed human CD4 mRNA. The authors conclude that the 3WJ/PG-RCA technique provides an excellent means to detect very low levels of RNA expression in samples such as clinical specimens.

Monday, February 6, 2012

Circular RNAs and exon scrambling

RNA-Seq techniques are well integrated into the study of cancer genetics and are useful in locating splice variants in genes that may be indicative of a malignant cell. In a recent study, Salzman et al. observed, using RNA-Seq analysis, that a large amount of spliced transcripts isolated from cancer cells are actually circular in nature. The results suggest that other splicing modes are possible and may be useful in differentiating cancerous from normal tissue types. So-called "scrambled exons", which are evident by splicing order errors in various mRNA species, are likely a result of the circularization of these transcripts.

To arrive at this conclusion, the authors initially isolated total RNA from bone marrow cells, removed rRNA using the Ribo-Zero™ Kit (Human/Mouse/Rat), prepared RNA-Seq libraries using the Illumina TruSeq RNA-Seq Sample Prep Kit, and sequenced the libraries on an Illumina GAIIx sequencer. Leukemia samples were sequenced together, while remission blood samples and normal bone marrow subpopulations were sequenced in separate runs. In another analysis, the authors used paired-end mapping ratios to determine the relative abundance of scrambled isoforms to normal linear isoforms. Each gene was tiled by dividing it into even-length bins of 200 bp. Data were validated by qRT-PCR.

The researchers further concluded that the scrambled splicing activity appears to occur most commonly in the cytoplasm. Four potential models for circularization were presented, using techniques that also demonstrated the presence of circular RNA splice variants using RNase R exoribonuclease.

ResearchBlogging.orgSalzman, J. et al. (2012). Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0030733