Small Amounts Of RNA Research Articles

Performance Evaluation of Real Time Polymerase Reaction Assay (SCODA) for Detection of SARS-CoV-2

In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. Real Time PCR is useful for detection and quantification of genetic constitution of pathogens. This technique amplifies a tiny DNA target million or billion times in such a way that it can be easily studied by scientists. Availability of highly sensitive and specific assay for the detection of SARS-Cov-2 and easy accessibility of such was necessary for early diagnosis and effective management of COVID-19 infection. The aim of this study was to evaluate the performance characteristic of SCODA. Validation of SCODA was performed using synthesized standards and clinical samples previously tested using a commercially approved COVID-19 RT-qPCR detection kit (LifeRiver). The assay showed a linearity of 98.2% on the ORF1ab target and 99.8% on the N-gene target. The sensitivity and specificity were both 100%. Analysis for the LoD95 produced 74.04 (CI: 25 - 1000) cp/μl on ORF1ab gene and 1.119 (CI: 1 - 1) cp/μl on N-gene target with a precision of CV ≤ 3%. SCODA showed high comparable performance in comparison with LifeRiver and other commercial COVID-19 RT-qPCR test kits.

Chromatin Dynamics in Digestive System Cancer: Commander and Regulator.

Digestive system tumors have a poor prognosis due to complex anatomy, insidious onset, challenges in early diagnosis, and chemoresistance. Epidemiological statistics has verified that digestive system tumors rank first in tumor-related death. Although a great number of studies are devoted to the molecular biological mechanism, early diagnostic markers, and application of new targeted drugs in digestive system tumors, the therapeutic effect is still not satisfactory. Epigenomic alterations including histone modification and chromatin remodeling are present in human cancers and are now known to cooperate with genetic changes to drive the cancer phenotype. Chromatin is the carrier of genetic information and consists of DNA, histones, non-histone proteins, and a small amount of RNA. Chromatin and nucleosomes control the stability of the eukaryotic genome and regulate DNA processes such as transcription, replication, and repair. The dynamic structure of chromatin plays a key role in this regulatory function. Structural fluctuations expose internal DNA and thus provide access to the nuclear machinery. The dynamic changes are affected by various complexes and epigenetic modifications. Variation of chromatin dynamics produces early and superior regulation of the expression of related genes and downstream pathways, thereby controlling tumor development. Intervention at the chromatin level can change the process of cancer earlier and is a feasible option for future tumor diagnosis and treatment. In this review, we introduced chromatin dynamics including chromatin remodeling, histone modifications, and chromatin accessibility, and current research on chromatin regulation in digestive system tumors was also summarized.

P-517 Validating reference gene expression stability in human ovarian follicles, oocytes, cumulus cells, ovarian medulla, and ovarian cortex tissue

Abstract Study question How does the expression stability of commonly used reference genes (RG) vary in different human ovarian cells and tissues? Summary answer The RG stability differed among human ovarian cell types and tissues, but an RG with high stability was identified for each cell and tissue type. What is known already The expression of RGs used to normalize RT-qPCR may vary between different tissues, cell types, and experimental conditions. Hence, selecting the most appropriate RGs is critical in any experimental design to interpret data generated by RT-qPCR with the best accuracy. Human ovarian cells are phenotypically very different and often only available in limited amounts. In several animal species RG expression stability has been validated in oocytes and other ovarian cells, however, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. Study design, size, duration This is an experimental study performed at a university hospital from January 2021 to September 2021. Participants/materials, setting, methods The freely available NormFinder software was used to analyze the expression stability of five commonly used RGs (GAPDH, B2M, RPLP0, ACTB, and PPIA) in human oocytes (n = 160), preantral follicles (n = 160), cumulus cells (n = 13), ovarian medulla (n = 8), and ovarian cortex tissue (n = 60). Samples were collected from 29 patients (aged 28 years on average; range 14–36) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Main results and the role of chance The Normfinder software identified ACTB as the best RG for oocytes and cumulus cells; and B2M for medulla tissue and isolated follicles. Overall, comparisons of the cycle threshold (Ct) values demonstrated a wide variation among the RGs within the same group of samples (P < 0.05), especially for oocytes and preantral follicles with normalized RNA. The genes ACTB and RPLP0 showed the highest levels of expression and PPIA the lowest levels of expression in all types of samples, except for cortex tissue, where PPIA had the highest level of expression and B2M the lowest. These results infer that different results could be obtained when using different RGs for data normalization. The combination of two RGs only marginally increased stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For cultured ovarian cortex culture, GAPDH or ACTB were found to be the most stable genes depending on culture conditions. Limitations, reasons for caution Since human oocytes are scarce and contain a small amount of RNA, only five RGs were evaluated. Moreover, only germinal vesicle (GV) oocytes and preantral follicles (60-80 µm) were tested. Future studies may validate other genes and investigate the effect of oocyte maturation and follicle growth on RG stability. Wider implications of the findings Our findings highlight the importance of validating RGs for each cell type or tissue and culture condition. Hence, our results can be of use as guidance for future studies involving gene expression analyses in human ovarian cells and tissues, including oocytes and preantral follicles. Trial registration number Not applicable

Abstract 979: Analysis of metabolic and genetic changes in human colorectal cancer and colorectal metastatic tissue by combined NanoString technology and mass spectrometry imaging (MSI)

Abstract Introduction: The development of normal colon tissue to carcinoma can progress through several pathways, which exhibit different clinical, pathological and genetic features. Increasing evidence indicates that tumor development and metastases are not solely driven by genetic alterations but also the tumor microenvironment which is associated with changes in metabolic processes. While many studies show the importance of gene expression profiling in cancer progression, little is known about how this correlates with altered lipid metabolism at metastatic sites within the peritoneal cavity. NanoString nCounter provides specific and sensitive genetic profiling data identifying up to 800 genes simultaneously from small amounts of RNA. For biomolecule analysis, MSI is a powerful tool as it enables the rapid identification of phenotype-dependent MS signatures from tissues with little sample preparation. The aim of this study was to investigate the characteristics of secondary tumors at different metastatic sites compared to the primary tumors using a combination of immunohistochemistry, NanoString analysis and Desorption Electrospray Ionization (DESI) MSI. Methods: 12 fresh-frozen human colorectal adenocarcinoma and metastases samples were cut into 12μm sections. DESI-MSI (Xevo G2-XS, Waters) was performed in both polarities with 50μm spatial resolution. SCiLS Lab (Bruker) software were used for data analysis. Histologically-stained (H&E) adjacent sections were used for microscopy. For genomic analysis, RNA was extracted using AllPrep DNA/RNA micro kit from 20μm sections. Following this, RNA abundance was measured using Qubit fluorimeter. Lastly, mRNA hybridisation, detection and scanning was performed on a NanoString nCounter mRNA Gene Expression system and analysed using nSolver 4.0 software. Results: H&E staining allowed the visualisation of different tissue histopathologies. These differences are echoed in selected DESI-MSI ion images. DESI spectra from distinct regions of the tissue show differences in biomolecule expression especially for phospholipids (m/z 600-900). Ions such as m/z 812 correlated with the non-cancerous region whereas others such as m/z 861 were only observed in the cancerous regions of the tissue. Gene expression analysis showed concordance between primary and metastatic tumors such as in RAS/RAF genes. Others, such as IL-6 expression was seen to upregulated in the metastatic sites compared to the primary tumor. Multivariate analysis enabled identification of biomolecules representative of different tissue phenotypes, with the data showing putative MS biomarkers associated with genetic mutations. Conclusion: Results confirmed the advantage of the combined techniques to reveal spatial correlations between changes in lipid metabolism, tumor heterogeneity and gene expression. Citation Format: Yasmin Shanneik, Emrys Jones, Michal Smiga, Bipasha Chakrabarty, Omer Aziz, Steven Pringle, Kaye Williams, Adam McMahon. Analysis of metabolic and genetic changes in human colorectal cancer and colorectal metastatic tissue by combined NanoString technology and mass spectrometry imaging (MSI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 979.

Clinical relevance of serum-derived exosomal messenger RNA sequencing in patients with non-Hodgkin lymphoma.

Background: The clinical utility of mRNA cargo in exosomes is unclear, although exosomes have potential as non-invasive biomarkers. This study aimed to investigate the feasibility of exosomal mRNA sequencing for monitoring disease status and predicting outcomes in non-Hodgkin lymphoma (NHL) patients.Methods: Exosomes were isolated from archived serum samples of 33 patients with NHL who were registered into our prospective cohort: diffuse large B-cell lymphoma (DLBCL, n = 17), intravascular B-cell lymphoma (IVL, n = 1), primary mediastinal large B-cell lymphoma (PMBL, n = 4), follicular lymphoma (FL, n = 3), mantle cell lymphoma (MCL, n = 3), and extranodal NK/T-cell lymphoma (ENKTL, n = 5). Exosomal mRNA sequencing was performed, and its concordance with clinical course was analyzed and compared with those of circulating tumor DNA (ctDNA) mutations.Results: Exosomal mRNA sequencing was performed successfully in 26 cases (79%, 26/33), whereas the remaining seven cases were not completed due to their small amount of RNA. The exosomal mRNA sequencing of DLBCL showed gene expression profiles consistent with activated B-cell-like and germinal center type. The longitudinal assessment of exosomal mRNA sequencing results in accordance with the clinical course showed that the post-treatment changes of exosomal mRNA expression were more consistent with treatment outcome than were those of ctDNA mutations. In particular, the exosomal mRNA expression of genes such as BCL2 and BCL6 was increased at the time of disease progression in DLBCL and FL patients.Conclusions: This study demonstrated the feasibility of exosomal mRNA expression profiles as a biomarker for NHL patients. Our results might provide the rationale for studies to explore the potential of exosomal mRNA as a biomarker in NHL patients.

Abstract 2283: Validation & application of next-generation sequencing panel YuanSu S for gene rearrangements/fusions detection

Abstract Introduction: Gene rearrangement/fusion play an important role in cancer diagnosis and treatment. Comparing with routine diagnostic methods, Sanger sequencing, fluorescence in situ hybridization (FISH) & immunohistochemistry (IHC), next-generation sequencing (NGS)-based panel can provide genetic information with robust sensitivity and precision. Moreover, panel-based RNAseq can detect novel fusions and only requires a small amount of RNA. We have developed a 450-gene DNA & RNA NGS panel, YuanSu S, for clinical sample detection. In this study, YuanSu S was applied to detect gene rearrangements/fusions and determine its limit of detection (LOD) and concordance with FISH/IHC, and difference between DNAseq & RNAseq. Methods: A total of 184 formalin fixed and paraffin embedded (FFPE) sarcoma samples were detected by YuanSu S at OrigiMed, a laboratory certified by College of American Pathologists (CAP) & Clinical Laboratory Improvement Amendments (CLIA). Gene fusions were analyzed by OriFusion, an OrigiMed's proprietary algorithm. For LOD determination, gradient amounts of RNA or mixed cell line samples were processed for sequencing. To confirm its concordance with FISH/IHC, 36 sarcomas FFPE samples were examined by FISH (33/36) or IHC (3/36). A cohort of 148 tumor samples was used to analyze rearrangement/fusion detection capability. Results: For RNAseq in YuanSu S, LOD was determined as 5 copies/1ng RNA input and was verified by ddPCR. Samples with rearrangement/fusion ≥ 5% detected by DNAseq were confirmed by ddPCR. For the interfering substance validation, results of samples treated with ethanol or necrotic tissue were consistent with untreated samples. Furthermore, two different Illumina platforms, Nextseq 500 & Novaseq 6000, showed no difference in sequencing abilities. 36 sarcoma FFPE samples were assessed by NGS and verified by FISH or IHC. All the 19 samples with FISH or IHC positive were detected the same rearrangements/fusions by YuanSu S, whereas the negative samples had no fusion detected. In the cohort of 148 samples, 426 rearrangements/fusions were detected by YuanSu S. 101 (23.7%) were detected both by DNAseq & RNAseq; 204 (47.9%) were determined by DNAseq only; while 121 (28.4%) were determined by RNAseq only. Rearrangements were detected by DNAseq in 97 (65.5%) samples, and 40 (41.2%) may benefit from FDA-approved drugs. Importantly, 51 (34.5%) samples had fusions detected only by RNAseq, and 33 (64.7%) may benefit from targeted therapies. Conclusion: Our results confirmed the reliability and advantages of YuanSu S, a DNA plus RNA NGS panel. Experience from YuanSu S indicated that integrative DNA & RNA sequencing could increase sensitivity of fusion detection. Therefore, the combination of DNAseq & RNAseq is able to screen more patients carrying fusion targets who could benefit from corresponding inhibitors and play an important role in guiding treatment decisions. Citation Format: Jiacheng Cai, Feng Li, Haiyan Wu, Weifeng Wang, Qiuyue Pan, Lin Zhang, Kai Wang. Validation & application of next-generation sequencing panel YuanSu S for gene rearrangements/fusions detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2283.

Longitudinal epitranscriptome profiling reveals the crucial role of N6-methyladenosine methylation in porcine prenatal skeletal muscle development

N6-methyladenosine (m6A) represents the most abundantly occurring mRNA modification and is involved in the regulation of skeletal muscle development. However, the status and function of m6A methylation in prenatal myogenesis remains unclear. In this study, we first demonstrated that knockdown of METTL14, an m6A methyltransferase, inhibited the differentiation and promoted the proliferation of C2C12 myoblast cells. Then, using a refined m6A-specific methylated RNA immunoprecipitation (RIP) with next generation sequencing (MeRIP-seq) method that is optimal for use with samples containing small amounts of RNA, we performed transcriptome-wide m6A profiling for six prenatal skeletal muscle developmental stages spanning two important waves of porcine myogenesis. The results revealed that, along with a continuous decrease in the mRNA expression of the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), the m6A methylome underwent highly dynamic changes across different development stages, with most of the affected genes being enriched in pathways related to skeletal muscle development. RNA immunoprecipitation confirmed that IGF2BP1 targets 76 genes involved in pathways associated with muscle development, including the key marker genes MYH2 and MyoG. Moreover, small interfering RNA (siRNA)-mediated knockdown of IGF2BP1 induced phenotypic changes in C2C12 myoblasts similar to those observed with knockdown of METTL14. In conclusion, we clarified the dynamics of m6A methylation and identified key genes involved in the regulatory network of porcine skeletal muscle development.

Predictive Immune Modeling of Solid Tumors.

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment response challenging. The ability to resolve the relative populations of immune cells present in and around the tumor tissue has been shown to be clinically-relevant to understanding response, but is limited by traditional techniques such as flow cytometry and immunohistochemistry (IHC), due the large amount of tissue required, lack of accurate cell type markers, and many technical and logistical hurdles. One assay (e.g., the ImmunoPrism Immune Profiling Assay) overcomes these challenges by accommodating both small amounts of RNA and highly degraded RNA, common features of RNA extracted from clinically archived solid tumor tissue. The assay is accessed via a reagent kit and cloud-based informatics that provides an end-to-end quantitative, high-throughput immuno-profiling solution for Illumina sequencing platforms. Researchers start with as few as two sections of formalin-fixed paraffin-embedded (FFPE) tissue or 20-40 ng of total RNA (depending on sample quality), and the protocol generates an immune profile report quantifying eight immune cell types and ten immune escape genes, capturing a complete view of the tumor microenvironment. No additional bioinformatic analysis is required to make use of the resulting data. With the appropriate sample cohorts, the protocol may also be used to identify statistically significant biomarkers within a patient population of interest.

Biogenesis, delivery, and function of extracellular RNA

The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA.

The RNA in reticulocytes is not just debris: It is necessary for the final stages of erythrocyte formation

Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

The molecular and pathway characterization of patients with metastatic castration resistant prostate cancer (mCRPC) refractory to therapy with abiraterone acetate or enzalutamide: Preliminary results from the SU2C/PCF/AACR West Coast Prostate Cancer Dream Team (WCDT).

79 Background: Progressive metastatic castration resistant prostate cancer (mCRPC) has historically been challenging to biopsy and characterize on a molecular basis because of its bone tropism. Since mechanisms of resistance to androgen signaling inhibitors such as enzalutamide or abiraterone are not fully understood, both an unbiased and a targeted assessment of the molecular landscape of these patients is required. Methods: Patients (pts) with mCRPC undergo biopsy at one of five West Coast Prostate Cancer Dream Team (WCDT) clinical sites, using a uniform biopsy protocol, following central radiologic review. Tissue is both frozen, and formalin fixed/paraffin embedded (FFPE). Frozen tissue undergoes laser capture microdissection (LCM) for RNA seq, DNA seq, and array comparative genomic hybridization (aCGH). An RNA seq process was developed that allows using extremely small quantities of RNA (approximately 1 ng). FFPE tissue undergoes a CLIA-certified assessment of a 37-gene mutational panel, FISH for AR, and IHC for PTEN. Peripheral blood is collected for miRNA, immune responses, and CTC analysis including aCGH. Pathway assessment integrating clinical, RNA seq, DNA seq, aCGH data is undertaken using PARADIGM analysis. Results: Thirty six of 300 planned mCRPC pts have undergone a metastasis biopsy: 17 from bone, eight from liver, one from lung, and 10 from distant lymph nodes. Tumor is present in around 75% of the frozen specimens. To date, LCM has been undertaken in 11 samples, with RNA seq done in six, DNA whole exome seq in one, aCGH in four. FFPE tissue has been evaluated by mutational panel sequencing (n=9), FISH for AR (n=11), and IHC for PTEN (n=13). CTC have been isolated in 33 pts. aCGH has been successfully undertaken in paired CTC and biopsy specimens. Expression data from patients with full RNA sequencing have been analyzed by PARADIGM, with top disrupted pathways identified. Conclusions: Biopsies of mCRPC, including from bony sites, can be undertaken and used for molecular and pathway analysis. Sufficient tissue for unbiased and targeted assessment can be obtained. Linkage of results from these studies to the clinical characteristics of these patients will reveal important insights into mechanisms of resistance.

Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.

Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.

Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation

BackgroundAnalyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting. With current high-speed sequencers, it is necessary to flank the cDNAs with specific adapters. Adding template-switching oligonucleotides to reverse transcription reactions is the most commonly used approach when working with very small quantities of RNA even from single cells.ResultsHere we compared the performance of DNA-RNA, DNA-LNA and DNA oligonucleotides in template-switching during nanoCAGE library preparation. Test libraries from rat muscle and HeLa cell RNA were prepared in technical triplicates and sequenced for comparison of the gene coverage and distribution of the reads within transcripts. The DNA-RNA oligonucleotide showed the highest specificity for capped 5′ ends of mRNA, whereas the DNA-LNA provided similar gene coverage with more reads falling within exons.ConclusionsWhile confirming the cap-specific preference of DNA-RNA oligonucleotides in template-switching reactions, our data indicate that DNA-LNA hybrid oligonucleotides could potentially find other applications in random RNA sequencing.

Feasibility of RNA sequencing to detect TMPRSS2:ERG fusions following laser capture microdissection of prostate cancer.

76 Background: Recent sequencing efforts continue to fuel our growing insight into the genetic basis of prostate cancer (PCa). One of the key challenges remaining is to identify genetic events that drive PCa biology. RNA sequencing (RNA-seq) is commonly used to determine unbiased gene expression as a proxy for tumor biology. However, tumors often contain a mixture of cancer and normal cells that can confound data analysis. Cancer cells can be isolated by laser capture microdissection (LCM), but the feasibility of RNA-seq on small amounts of RNA following LCM of PCa tissue needs to be assessed. Methods: A set of 5 localized PCa specimens, frozen at the time of prostatectomy, was analyzed. RNA was isolated following LCM from these 5 specimens and assessed for quality with an Agilent Bioanalyzer and for quantity with RiboGreen. Paired-end, 300-bp insert sequencing libraries were prepared from the 5 specimens in duplicate; one specimen was also prepared for sequencing without LCM. The 12 samples were then sequenced on a single lane of an Illumina HiSeq. Sequencing reads were aligned with Tophat to build hg19 of human genome. Initial quality control was performed with Picard using refFlat and ribosomal RNA (rRNA) annotations provided by UCSC. PCR using previously published primer pairs was used to confirm the presence of TMPRSS2:ERG fusions. Results: 4-5mm2 of tumor was targeted by LCM from each PCa specimen. At least 100ng of total dsDNA was generated for library construction. For the first run, an average of 8,743,722 reads per paired-end was obtained (range: 1,335,939 to 16,134,097); the second run was comparable (mean: 8,612,188 reads; range: 1,312,823 to 15,842,480). Mapping of the sequences revealed no 5’ or 3’ read coverage bias, but the percentage of mappable reads per sample was less than 30%; sequencing was inefficient due to a high percentage of rRNA (mean: 38% per sample; range 28 to 44%). However, TMPRSS2:ERG fusions were confirmed in the samples with known fusion-positive status. Conclusions: RNA-seq is feasible and can detect fusions that are known and highly expressed. The inclusion of rRNA reduces the depth of coverage of the transcriptome and additional optimization is required to maximize efficiency.

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

BackgroundWith lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles.ResultsRNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts.ConclusionsAn efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Distribution of angiotensin II receptor expression in the microcirculation of striated muscle.

The current study was undertaken to localize and identify angiotensin II (Ang II) receptor subtypes in the microcirculation of striated muscle. Cremaster muscles from 7- to 8-week-old Sprague-Dawley rats were excised, placed in a dissection solution maintained at 4 degrees C, and 3 branch orders of arterioles and venules, as well as capillaries and a muscle specimen, were microdissected under a stereomicroscope. Reverse transcription polymerase chain reaction (RT-PCR) methods were developed for purification and amplification of extremely small amounts of RNA (<5 ng/microL) from whole tissue samples. RNA was isolated from each sample, reverse transcribed, and the cDNA products were amplified by polymerase chain reactions (PCR) specifically primed with either AT(1a), AT(1b), or AT(2) receptor primers. The products were electrophoretically size-fractionated on an agarose gel, stained with ethidium bromide to visualize DNA bands, and analyzed to determine the presence or absence of AT receptor subtypes. Protein expression was confirmed by Western blotting pooled samples with specific antiserum. AT(1a) and AT(2) receptors were found in nearly all orders of both arterioles and venules, as well as the skeletal muscle biopsies. AT(1b) receptors, if present, were only observed on a few instances in the arterioles. Furthermore, PCR reactions specifically primed for skeletal muscle cell (MHC(2B)) and endothelial cell (eNOS) specific proteins demonstrated that there was no cross-contamination between the vessels and the skeletal muscle biopsies. This study describes a unique method for the isolation and preparation of microvessels and provides the first data directly demonstrating the presence of AT(1) and AT(2) receptors in microvessels as well as in skeletal muscle fibers.

Transcribed-ultra conserved region expression profiling from low-input total RNA

BackgroundUltra Conserved Regions (UCRs) are a class of 481 noncoding sequences located in both intra- and inter-genic regions of the genome. The recent findings that they are significantly altered in adult chronic lymphocytic leukemias, carcinomas, and pediatric neuroblastomas lead to the hypothesis that UCRs may play a role in tumorigenesis.ResultsWe present a novel application of Ribo-SPIA™ isothermal linear amplification of minute RNA quantities for quantifying Transcribed-UCR (T-UCR) expression by quantitative PCR. Direct comparison of non-amplified with amplified cDNA in two neuroblastoma cell lines showed that the amplification approach increases sensitivity and repeatability in T-UCR quantification. It is noteworthy that the Ribo-SPIA™ step allowed us to analyze all 481 T-UCRs by using 150 ng of RNA, while introducing a minimal bias and preserving the magnitude of relative expression. Only the less abundant T-UCRs have high intra-assay variability, consistently with the Poisson distribution statistics and stochastic effects on PCR repeatability.ConclusionsWe demonstrated that the quantification procedure shown here is an accurate and reliable technique for genome-wide non coding gene (i.e., UCRs) profiling using small amounts of RNA. This issue is particularly important because studies of transcription regulation are increasingly conducted in small homogeneous samples, such as laser capture microdissected or sorted cell populations.

Multiple Gene Expression Analyses in Human Lymphoid Tissues by TaqMan Low-density Array Using Amplified RNA Isolated From Paraffin-Embedded Samples

In this study, we examined whether small amounts of RNA available from fixed and paraffin-embedded samples could be increased and quantified while conserving the gene expression profile. To this aim, RNA was extracted by a modified version of an earlier described method and was amplified with a linear amplification system. Furthermore, using TaqMan low-density arrays technology, the expression of multiple genes was correlated in matched snap-frozen and paraffin-embedded tissues. The entire technical procedure was assessed on 7 reactive lymph nodes. The results of the study showed that highly degraded RNA could be amplified to a mean of a 1200 to 3500-fold increase. Furthermore, the amount of cDNA and the polymerase chain reaction amplification efficiency was higher in amplified RNA than in unamplified RNA (89% vs. 82%). The analysis of Ct and DeltaCt values showed strong correlations in matched amplified versus unamplified, fixed and frozen samples, thus demonstrating the conservation of gene expression in amplified RNA. We conclude that small amounts of RNA from paraffin-embedded tissues can be successfully amplified without altering the gene expression. Consequently, TaqMan low-density array technology could be used successfully for the analysis of archival fixed and paraffin-embedded lymphoid tissue.

RT-PCR detection of HIV in Republic of Macedonia.

The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works. The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5). ELFA test for combined detection of HIV p24 antigen and anti HIV-1+2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity. Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly. In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is fast, simple for manipulation; with low detection level of 60 IU/ml. RT-PCR needs a small amount of RNA, as well as a small volume of sample. HIV RNA was detected in different periods of time with different clinical presentations in patients, with or without antiretroviral therapy. RT-PCR method gives the opportunity for reliable determination of HIV-1 RNA with border of detection of 60 IU/ml. The test is reproducible and has high analytical and clinical specificity.