Aiguo Zhang

Small Interfering RNA and Gene Expression Analysis Using a Multiplex Branched DNA Assay without RNA Purification

The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1α (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes’ expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.

A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma

A simple and accurate method for measuring the biological effects of radiation is of increasing importance, especially in mass casualty scenarios. We have therefore developed a new biodosimetric technique targeting circulating B1 DNA in mouse plasma by branched DNA signal amplification for rapid quantification of plasma DNA. This technology targets repetitive elements of the B1 retrotransposon in the mouse genome, followed by signal amplification using Panomics Quantigene 2.0 reagents. Evaluation was conducted concerning precision, accuracy and linearity. Plasma samples were collected from mice 0-24 h after 0-10 Gy total body irradiation (TBI). The average inter- and intra-assay coefficients of variance were 8.7% and 12.3%, respectively. The average recovery rate of spiked DNA into plasma was 89.5%. This assay revealed that when BALB/c and NIH Swiss mice were exposed to 6 Gy TBI, plasma B1 DNA levels increased significantly at 3 h post-TBI, peaked at 9 h and gradually returned toward baseline levels in 24 h. A dose-dependent change in plasma DNA was observed at 9 h post-TBI; the dose-response relation was monotonic, exhibiting linearity for BALB/c mice from 3 to 6 Gy (r = 0.993) and NIH Swiss mice from 3 to 7 Gy (r = 0.98). This branched DNA-based assay is reliable, accurate and sensitive in detecting plasma B1 DNA quantitatively. A radiation dose-correlated increase in plasma B1 DNA was demonstrated in BALB/c and NIH Swiss mice in the dose range from 3 to 6 Gy, suggesting that plasma B1 DNA has potential as a biomarker for radiation biological effect.

A basic fibroblast growth factor analog for protection and mitigation against acute radiation syndromes.

AbstractThe effects of fibroblast growth factors and their potential as broad-spectrum agents to treat and mitigate radiation injury have been studied extensively over the past two decades. This report shows that a peptide mimetic of basic fibroblast growth factor (FGF-P) protects and mitigates against acute radiation syndromes. FGF-P attenuates both sepsis and bleeding in a radiation-induced bone marrow syndrome model and reduces the severity of gastrointestinal and cutaneous syndromes; it should also mitigate combined injuries. FGF‐2 and FGF-P induce little or no deleterious inflammation or vascular leakage, which distinguishes them from most other growth factors, angiogenic factors, and cytokines. Although recombinant FGFs have proven safe in several ongoing clinical trials, they are expensive to synthesize, can only be produced in limited quantity, and have limited shelf life. FGF-P mimics the advantageous features of FGF‐2 without these disadvantages. This paper shows that FGF-P not only has the potential to be a potent yet safe broad-spectrum medical countermeasure that mitigates acute radiotoxicity but also holds promise for thermal burns, ischemic wound healing, tissue engineering, and stem-cell regeneration.Health Phys. 106(6):000-000; 2014

Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived “driver” and “drug-resistant” alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called “liquid biopsy” allows for the dynamic monitoring of the patients’ tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

Abstract 1503: Rapid detection of somatic mutations in cancer genes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In the context of personalized cancer treatment, tumor mutational analysis by DNA sequencing has been described frequently as the “gold standard”, yet DNA sequencing is one of the least sensitive methods for characterizing mutation. At least 10-20% of a DNA population must be mutant for the mutation to be detected by standard DNA sequencing. Implicit acceptance that this level of sensitivity is sufficient to characterize tumor mutations only makes sense if one assumes that tumors are monoclonal and that all the critical driver mutations will be present in the bulk of the tumor. Yet these are incorrect assumptions. In fact, it has become clear that most if not all colon tumors are polyclonal and heterogeneous. Consequently, tumor driver mutations may not be detectable using standard DNA sequencing methods. In order to rapidly detect these low frequency mutations in tumor derived DNA we have developed a new technology that allows the detection of tumor driver and drug resistance somatic mutations at <0.1% sensitivity using quantitative PCR (qPCR) without the need for digital PCR or droplet digital PCR (ddPCR) methodology. We utilize xeno-nucleic acid (XNA) clamping probes that are specific for the target gene wild-type template and allow only the selective amplification of mismatched mutant templates. Direct detection of driver and drug resistance mutations within 2 hours of obtaining samples in KRAS, NRAS, BRAF, EGFR and JAK2 utilizing real-time qPCR with interchelating fluorescence dye detection. As well as a new end-point post-PCR microtiterplate based target gene mutant amplicon capture format that utilizes colorimetric or chemiluminescence detection will be described. Citation Format: Michael J. Powell, Larry Pastor, Rachel Diaz, Lily Chen, George Wu, Claudia Li, Aiguo Zhang. Rapid detection of somatic mutations in cancer genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1503. doi:10.1158/1538-7445.AM2014-1503