RNA Extraction Efficiency Research Articles

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells.

Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.

Valine-Induced Isoleucine Starvation in Escherichia coli K-12 Studied by Spike-In Normalized RNA Sequencing.

Escherichia coli cells respond to a period of famine by globally reorganizing their gene expression. The changes are known as the stringent response, which is orchestrated by the alarmone ppGpp that binds directly to RNA polymerase. The resulting changes in gene expression are particularly well studied in the case of amino acid starvation. We used deep RNA sequencing in combination with spike-in cells to measure global changes in the transcriptome after valine-induced isoleucine starvation of a standard E. coli K12 strain. Owing to the whole-cell spike-in method that eliminates variations in RNA extraction efficiency between samples, we show that ribosomal RNA levels are reduced during isoleucine starvation and we quantify how the change in cellular RNA content affects estimates of gene regulation. Specifically, we show that standard data normalization relying on sample sequencing depth underestimates the number of down-regulated genes in the stringent response and overestimates the number of up-regulated genes by approximately 40%. The whole-cell spike-in method also made it possible to quantify how rapidly the pool of total messenger RNA (mRNA) decreases upon amino acid starvation. A principal component analysis showed that the first two components together described 69% of the variability of the data, underlining that large and highly coordinated regulons are at play in the stringent response. The induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS, and Lrp regulons. This underlines the notion that with the high resolution possible in deep RNA sequencing analysis, any different starvation method (e.g., nitrogen-deprivation, removal of an amino acid from an auxotroph strain, or valine addition to E. coli K12 strains) will produce measurable variations in the stress response produced by the cells to cope with the specific treatment.

Investigation into the presence of Hepatitis E Virus in commercially available venison charcuterie in Scotland

Hepatitis E Virus is an RNA virus of the Hepeviridae family that affects hepatocytes and seriously impacts the health of immunocompromised patients. Genotypes 3 and 4 have been circulating in animal populations such as swine and deer in developed countries, causing food borne infections associated with consumption of contaminated animal food products. While HEV has been circulating in European deer populations and causing food borne infections in Asia, its presence in Scottish venison food products has not been investigated. This study assesses 4 ready-to-eat venison charcuterie products for HEV by performing RNA extraction on duplicate samples with a Qiagen RNeasy Midi kit and using Mengovirus as a process control to determine virus recovery by RT-qPCR. This was followed by RT-PCR with previously described HEV primers, and ligation of the suspected positive fragments in a recombinant pGEMT Easy Vector system which was then cloned in competent E. coli cells for propagation. Recombinant plasmids were isolated and subjected to Sanger sequencing. An average RNA extraction efficiency of 5.02% was achieved across all samples, but large deviations between duplicates suggests that further optimisation is necessary in RNA extraction from food products with complex matrices. HEV-sized PCR fragments were detected in 3 out of 4 food products, but the sequencing data showed inefficient ligation of the fragments in the vector, possibly due to low RNA amount and excess of dimerised primers. The likelihood of HEV presence in Scottish venison needs to be investigated further for the detection of transmission routes and the implementation of improved surveillance policies.

Abstract 2807: Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads

Abstract Background: Multicolor flow cytometry has provided deep characterization of tumor-infiltrating lymphocytes (TILs). Routine fluorescence-activated cell sorting (FACS) methods discard cells after counting, precluding transcriptome analysis, which is key to understanding the activation states of immune cells. FACS-sorted cells pose 3 problems for transcriptomics: low cell count, cell rupture from high-pressure sort process, and inefficient centrifugation and recovery of cells from large collection volumes. SMART-Seq (TaKaRa/Clontech) is a highly sensitive, reverse transcriptase-based method to amplify low-input RNA that is commonly used to generate RNA-Seq libraries. After FACS, cell RNA can be purified by Qiagen RNeasy or nucleic acid binding solid-phase reversible immobilization (SPRI) magnetic beads. Alternatively, cells can be spun down after FACS then directly lysed as input to the SMART-Seq method. We sought to compare different methods and design a high-throughput protocol to capture the transcriptome from FACS-sorted, rare immune cell types. Methods: A dilution series of 50-5000 human T cells was generated to test efficiency and sensitivity of RNA extraction. We compared RNA extraction yields by column (Qiagen RNeasy Low Input Micro Kit) or SPRI bead (Agencourt RNA-Advanced Cell v2, Beckman Coulter). Direct lysis of cells in SMART-Seq buffer was tested as an alternative to extraction. RNA yields were quantified by High Sensitivity Agilent Bioanalyzer or digital droplet PCR (BioRad). Extracted RNA was reverse transcribed and amplified using SMART-Seq v2 followed by library preparation (Nextera XT DNA kit) and sequencing (NovaSeq Illumina). To obtain TILs, mice were implanted subcutaneously with MC38 tumor cells, then tumors were harvested, disassociated, and FACS sorted to isolate T cells (CD4+, CD8+) and macrophages (yields ranged from 1000-8000 cells). Results: Direct lysis followed by SMART-Seq was found to work well with diluted T cells; however, the method was sensitive to large buffer volumes and was unreliable for small numbers of pelleted, FACS-sorted cells (<5000). SPRI beads provided higher yields than Qiagen columns and were amenable to plate-based automation. RNA-Seq analysis of a T cell dilution series showed high concordance between SPRI and Qiagen columns, although more genes expressed at very low levels were detected in SPRI-purified samples. We used the SPRI bead method to characterize the transcriptome of TILs from implanted mouse tumors. Expected patterns of immune marker expression by immune cell type were observed. Conclusions: Use of SPRI beads to extract RNA from FACS-sorted immune cells was sensitive and reproducible, enabling transcriptomic characterization of <5000 cells. Successful profiling of TILs derived from mouse syngeneic tumors suggests that the method can be used to probe activation states of rare immune cell types in the tumor microenvironment. Citation Format: Aiqing He, Manling Ma-Edmonds, Chan Gao, Vishal Patel, David Galbraith, Yan Zhang, Qi Guo, Heshani DeSilva, Gennaro Dito, Amy Truong, Sunil Kuppasani, Kandasamy Ravi, Omar Jabado. Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2807.

Preparation of iron oxide silica particles for Zika viral RNA extraction

In this work, a robust synthetic pathway for magnetic core preparation and silica surface coating of magnetic microparticles is presented. Silica-coated magnetic particles are widely used to extract DNA and RNA from various biological samples. We present a novel route for the synthesis of iron oxide silica particles (Fe3O4@Silica) and demonstrate their performance for extracting ZIKA viral RNA from serum. The iron (II, III) oxide (Fe3O4), magnetite core is first prepared by ammonia neutralization of ferrous and ferric chloride aqueous solution under argon, followed by the addition of citrate salt to stabilize the surface of the resultant magnetic nanospheres. After this one-pot, two-step synthesis, the magnetic nanospheres are consumed during silica coating by hydrolysis of tetraethoxysilane (TEOS) under alkaline condition. The final product is a sphere-like magnetic aggregate with a size range of 1–2 micron. By simply suspending the magnetic aggregates in guanidinium chloride solution, the silica surface can be prepared for RNA binding. The RNA extraction efficiency was evaluated by extracting ZIKA viral RNA from serum followed by a PCR-based assay. The data indicate excellent recovery of target RNA and removal of PCR inhibitors. This manufacturing procedure for the silica coated microparticles provides a low-cost, effective and ready for scale-up method whose performance is equivalent to commercial alternatives such as magnetic silica surface particles for DNA and RNA sample preparations. The cost of the clinical assays could be largely decreased due to the 100 fold reduction in cost by replacing the commercially available magnetic particles with the developed material for RNA extraction.

Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples

Human enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3–18 RNA copies/µL for the targeted human pathogenic viruses and 20–40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination.

A High-Throughput RNA Extraction for Sprouted Single-Seed Barley (Hordeum vulgare L.) Rich in Polysaccharides.

Germinated seed from cereal crops including barley (Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the excess polysaccharides. Here, we compare the current methods for RNA extraction applicable to germinated barley tissue. We found that although some of these standard methods produced high-quality RNA, the process of extraction was drastically slow, mostly because the frozen seed tissue powder from liquid N2 grinding became recalcitrant to buffer mixing. Our suggested modifications to the protocols removed the need for liquid N2 grinding and significantly increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat.

Abstract 4285: Intra-individual variation in circulating miRNA expression levels in human plasma samples

Abstract Circulating miRNAs as possible non-invasive biomarkers for disease risk assessment and cancer early diagnosis has attracted increasing interest. Little information, however, is available regarding the intra-individual variation of circulating miRNA levels. We measured miRNA expression in repeated plasma samples that were collected 6 to 12 months apart from 53 healthy individuals participating in a sub-study nested in the Shanghai Women's and Shanghai Men's Health Studies. Total RNA, including miRNA, was isolated from plasma using Qiagen's miRNeasy Serum/Plasma Kit. Three synthetic spike-in RNA oligos (osa-miR414, cel-miR248, and ath-miR159a) were added to control for variances in the starting material and the efficiency of RNA extraction. NanoString's Human v2 miRNA Expression Assay, which includes CodeSets of probes specific for 800 common human miRNAs, was used for the miRNA assays. The derived miRNA counts were first corrected by subtracting the background counts and then were normalized by using the average count from three spike-in oligos and the top fifty miRNAs that gave most significant hybridization signals. The intra-individual variation was evaluated by an intra-class correlation coefficient (ICC). A total of 185 miRNAs were detected in at least 10% of samples. The detectable rate varied for each miRNA, with 69 and 44 miRNAs being detected in 50% and 75% of samples, respectively. The median ICC was 0.46 for all of the 185 detectable miRNAs. Among them, 41% (75 miRNAs) had an ICC ≥0.5, and 42 miRNAs had an ICC ≥0.6. The ICC is higher for miRNAs with higher expression levels and higher detection rates, when compared to their counterparts with lower expression or detection rates. In summary, we found that 185 miRNAs were expressed in at least 10% of plasma samples, and 69 miRNAs in 50% of samples from healthy Chinese men and women, and that 75 of these miRNAs had an ICC ≥0.5. These results suggest that common circulating miRNAs can serve as reliable biomarkers for epidemiological and clinical research. Citation Format: Jie Wu, Hui Cai, Yong-Bing Xiang, Charles Matthews, Fei Ye, Wei Zheng, Qiuyin Cai, Xiao-Ou Shu. Intra-individual variation in circulating miRNA expression levels in human plasma samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4285.

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses. Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 °C.

Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts

Molecular detection assays are increasingly becoming routine diagnostic techniques for bacterial infection; however, their sensitivities are restricted by the low concentrations of bacteria in clinical samples. Here, we report a new paradigm for ultrasensitive detection of bacteria. The principle of this approach is that by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts, assay sensitivity can be greatly improved. First, signature genes with abundant transcripts were screened by RNA-Seq. We confirmed that RT-PCR efficiently amplifies both DNA and RNA, while PCR amplifies only DNA. Unexpectedly, we found that the RNA extraction efficiency is relatively low, while simplified denaturation was more appropriate for transcript detection. For highly transcribed genes, RT-PCR consistently generated lower cycle threshold (Ct) values than those of PCR. The sensitivity of RT-PCR targeting abundant transcripts could detect quantities as low as one bacterium, which was not possible using PCR. Amplification of different genes among several other common bacteria also confirmed that transcript detection by RT-PCR is more sensitive than is DNA detection by PCR. Therefore, abundant transcript detection represents a universal strategy for ultrasensitive detection of bacteria.

等速電気泳動を用いた一細胞スケールRNA抽出における効率の評価

We report our efforts on evaluating the RNA extraction efficiency via microfluidic ITP (isotachophoresis). We have developed a theoretical model based on second-order kinetics and validated it with experimental results to explore the effect of the key ITP parameters, including target initial concentration, forward and reverse reaction constants, the maximum surface adsorption, ITP zone velocity and electric field. We also report that the polydimethylsiloxane microchannels are compatible with ITP-aided RNA extraction and yields 87.2 ± 9.4% (S. E.) extraction efficiency at single cell level.

RT-qPCR-based quantification of small non-coding RNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate messenger RNA (mRNA) translation into protein. MiRNAs play a key role in gene expression regulation, and their involvement in disease biology is well documented. This has fueled the development of numerous tools for the quantification of miRNA expression levels. These tools are based on three technologies: (microarray) probe hybridization, RNA sequencing, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In this chapter, we describe a quantification system based on RT-qPCR technology, which is currently considered as the most sensitive, flexible, and accurate method for quantification of not only miRNA but also RNA expression in general. To this purpose, we have divided the protocol in three sections: reverse transcription (RT) reaction, optional preamplification (PA), and finally qPCR. Three quality-control (QC) steps are implemented in this workflow for assessment of RNA extraction efficiency, sample purity (e.g., absence of inhibitors), and inter-run variations, by examining the detection level of different spike-in synthetic miRNAs. We conclude by demonstrating raw data preprocessing and normalization using expression data obtained from high-throughput miRNA profiling of human RNA samples.

Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine

Micro RNAs (miRNAs) have been shown to circulate in biological fluids and are enclosed in vesicles such as exosomes; they are present in urine and represent a noninvasive methodology to detect biomarkers for diagnostic testing. The low abundance of RNA in urine creates difficulties in its isolation, of which exosomal miRNA is a small fraction, making downstream RNA assays challenging. Here, we investigate methods to maximize exosomal isolation and RNA yield for next-generation deep sequencing. Upon characterizing exosomal proteins and total RNA content in urine, several commercially available kits were tested for their RNA extraction efficiency. We subsequently used the methods with the highest miRNA content to profile baseline miRNA expression using next-generation deep sequencing. Comparisons of miRNA profiles were also made with exosomes isolated by differential ultracentrifugation methodology and a commercially available column-based protocol. Overall, miRNAs were found to be significantly enriched and intact in urine-derived exosomes compared with cell-free urine. The presence of other noncoding RNAs such as small nuclear and small nucleolar RNA in the exosomes, in addition to coding sequences related to kidney and bladder conditions, was also detected. Our study extensively characterizes the RNA content of exosomes isolated from urine, providing the potential to identify miRNA biomarkers in human urine.

Clinical translation of human microRNA 21 as a potential biomarker for exposure to ionizing radiation

This study investigated to what extent the serum microRNA 21 (miR-21) level alters in response to ionizing radiation (IR). Initially, we evaluated the appropriateness of our RNA extraction efficiency and microRNA assay in serum, and then investigated the serum miR-21 level in 4 patients with breast cancer in 4 stages: pre- and postoperation, at the beginning radiotherapy, and after 25 sessions of radiotherapy with a total of 50 Gy irradiation, as well as in 20 healthy volunteers. The initial analysis showed the appropriateness of our RNA extraction efficiency and microRNA assay in serum for identifying people exposed to IR. We then analyzed the serum miR-21 level in another group of 40 patients with breast cancer before and after radiotherapy. During our large-scale analysis, the miR-21 level before radiotherapy was comparable with healthy volunteers (P = 0.10) and increased significantly after radiotherapy (P < 0.001)-an indication that this could discriminate irradiated patients from nonirradiated ones with high specificity (75%) and sensitivity (80%). According to this study, serum miR-21 has the potential to be used as a biomarker for the identification of people exposed to ionizing radiation.

Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA

Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. The PVL quantification procedure was optimized by inclusion of an exogenous control hepatitis C virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660, and the LTR region of SIVagmSAB were also optimized. Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV RNA in the same samples using the 'industry standard' method of branched-DNA (bDNA) signal amplification. Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs.

Efficiency of RNA extraction from selected bacteria in the context of biogas production and metatranscriptomics

Understanding the microbial population in anaerobic digestion is an essential task to increase efficient substrate use and process stability. The metabolic state, represented e.g. by the transcriptome, of a fermenting system can help to find markers for monitoring industrial biogas production to prevent failures or to model the whole process. Advances in next-generation sequencing make transcriptomes accessible for large-scale analyses. In order to analyze the metatranscriptome of a mixed-species sample, isolation of high-quality RNA is the first step. However, different extraction methods may yield different efficiencies in different species. Especially in mixed-species environmental samples, unbiased isolation of transcripts is important for meaningful conclusions. We applied five different RNA-extraction protocols to nine taxonomic diverse bacterial species. Chosen methods are based on various lysis and extraction principles. We found that the extraction efficiency of different methods depends strongly on the target organism. RNA isolation of gram-positive bacteria was characterized by low yield whilst from gram-negative species higher concentrations can be obtained. Transferring our results to mixed-species investigations, such as metatranscriptomics with biofilms or biogas plants, leads to the conclusion that particular microorganisms might be over- or underrepresented depending on the method applied. Special care must be taken when using such metatranscriptomics data for, e.g. process modeling.

Rapid extraction of total RNA from an anaerobic sludge biocoenosis

In order to assess the activity of metabolic pathways during anaerobic biogas production, it is necessary to isolate total RNA from the anaerobic sludge. mRNA activity profiling complements the quantification of excreted metabolites for a comprehensive anaerobic digestion model (ADM1). Four non-commercial total RNA extraction protocols were examined to extract total RNA from suspended solids of anaerobic sludge. The most suitable protocol was identified and optimised. In relation to total RNA extraction efficiency, total RNA purity and RNA integrity, the best homogenisation method was a combined method of nitrogen grinding and bead beating. When bead beating or nitrogen grinding was used alone for homogenisation, total RNA extraction efficiency was lower than when both homogenisation methods were applied. Depending on the homogenisation method, the whole RNA extraction procedure takes approximately 2 to 3 h, which is as fast as when using commercial available soil RNA extraction kits. The proposed method is rapid in extracting total RNA from a biocoenosis present in an anaerobic sludge environment. Furthermore, we could apply any of the extracted homogenization methods for reverse transcription and subsequent PCR amplification of the gene for the methyl coenzyme M reductase alpha subunit (mcrA/mrtA).

Importance of Suitable Reference Gene Selection for Quantitative RT-PCR during ATDC5 Cells Chondrocyte Differentiation

Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.

Improved RNA Extraction Method Using the BioMasher and BioMasher Power-Plus

The BioMasher is a disposable homogenizer that was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy diagnosis. Capable of preventing the biohazard risk from infectious samples, it also prevents cross-contamination among samples. The BioMasher is thus widely used in biochemical research, especially for RNA extraction. Here, we tested a novel BioMasher application for RNA extraction from animal and plant tissues. We also developed a grinding machine specific for the BioMasher, named the BioMasher Power-Plus. We developed RNA extraction protocols using the BioMasher combined with the BioMasher Power-Plus. We compared RNA extraction efficiency of the BioMasher with that of the FastPrep and the glass homogenizer. Though the RNA extraction efficiency by the BioMasher was nearly equivalent to that of the FastPrep and the glass homogenizer, sample preparation time was shorter for the BioMasher. The utility of RNA extraction by the BioMasher was examined in mouse, rat, and tomato tissue samples. In the rodent tissues, the highest extraction efficiency of total RNA was from liver, with lowest efficiency from fibrous tissues such as muscle. The quality of extracted total RNA was confirmed by agarose gel electrophoresis which produced highly visible clear bands of 18S and 28S rRNAs. Reproducibility among different operators in RNA extraction from tomato roots was improved by using the BioMasher Power-Plus. The BioMasher and BioMasher Power-Plus provide an effective and easy homogenization method for total RNA extraction from some rodent and plant tissues.

MicroRNA-155 As a Potential Plasma Biomarker for Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia,

Abstract 3669 BackgroundMicroRNAs (miRNAs) are short, non-coding RNA molecules that average 18–24 nucleotides long and act as important post-transcriptional regulators. It has been demonstrated that miRNAs stably circulate in the blood and can be detected in plasma and serum. Numerous disease states, including both solid tumor and hematologic malignancies, have been shown to release miRNAs into the circulation. Furthermore, these tumor cells possess their own characteristic expression patterns of miRNAs. This presents an opportunity for specific miRNAs to serve as biomarkers for different diseases. Previous studies have shown microRNA-155 (miR-155) to be elevated in low grade lymphomas such as Chronic Lymphocytic Leukemia (CLL) and Waldenstrom Macroglobulinemia (WM). Currently, an established biomarker for diagnostic or prognostic applications does not exist for these diseases. In our study, we investigate miR-155 as a potential biomarker for CLL and WM in plasma. MethodsBoth plasma and serum samples were collected from CLL patients, WM patients, and healthy individuals. In order to quantify miR-155 levels, 100 μl of each sample went through a round of RNA isolation, reverse transcription (RT) reaction, and real-time quantitative PCR (qPCR) by using miRNeasy mini kit (Qiagen), Taqman microRNA reverse transcription kit (Applied Biosystem) and RT2 Fast SYBR® Green/ROX qPCR Master Mixes (SABiosciences). Based on the RT-qPCR results, samples were classified as having either detectable miR-155 (Ct value less than 40) or undetectable miR-155 (Ct value more than 40). The ratio of detectable to undetectable samples was used to compare groups of samples: i) CLL plasma, ii) CLL serum, iii) WM plasma, iv) WM serum v) Normal plasma, and vi) Normal serum. To adjust for variations in efficiency of RNA extraction among samples, C. elegans synthetic microRNA-39 was added specifically after denaturation of the sample and used as a spiked-in control. By using the CD63 antigen as a marker, exosomes in the plasma and serum were also detected through flow cytometry. ResultsA total of 20 CLL plasma samples showed a 100% positive ratio of detectable miR-155 while 20 CLL serum samples had only a 50% positive ratio. In addition, a total of 96 WM plasma samples had a positive ratio of 74% while 30 WM serum samples showed a much lower positive ratio of 3%. Of these 96 samples, 13 were obtained from upfront WM patients while 83 were from relapsed/refractory WM patients. The 10 plasma and 10 serum samples from healthy individuals were all negative for miR-155. Lastly, preliminary data indicated that a higher level of exosomes may exist in plasma compared to serum. SummaryThis finding suggests that miR-155 in peripheral blood plasma could potentially be used as a clinical biomarker for WM and/or CLL. The considerable difference in detectable miR-155 between patient plasma and serum was unexpected. Reasons for this disparity are still under investigation. Our preliminary data indicated that this may be due to a difference in exosomes, which are known to carry miRNAs, between plasma and serum. Ultimately, further investigation is warranted within a larger cohort to confirm the status of miR-155 as a reliable plasma biomarker for these low grade B cell malignancies. Disclosures:Roccaro:Roche. Ghobrial:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.