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For example, we first collect all the RNA contents from a mammalian cell line. Say, for instance, we collect 80 uL of RNA with a concentration of 150 ng/uL. Next, to make cDNA, we take 1,000 ng of the RNA. However, this 1,000 ng is a completely random sampling of the total RNA we had. How is that random sample of RNA representative of the RNA contents originally in our cells?
It isn't really clear exactly what the questioner is asking, but…
… one way of thinking about this is to ask how likely is it that a low-abundance RNA will be absent from the sample taken from the preparation of RNA. This would make the sample non-representative at the qualitative level.
The question states that 12,000 ng of RNA is isolated. Since nothing else is specified I assume this is total RNA. A rough estimate of total RNA per eukaryotic cell is 20 pg.
This means that the preparation came from 6 x 105 cells. (I'm assuming 100% yield, but this won't really affect the conclusion.)
Now assume that a low-abundance mRNA is present at 10 copies per cell. The total RNA preparation therefore contains 6 x 106 copies of that low-abundance mRNA.
We take a sample of 1,000 ng from the total 12,000 ng. In other words we take 1/12th of the preparation of RNA. If that RNA preparation contains 106 copies of the low abundance mRNA how likely is it that we happen to take a sample which contains no copies of that low abundance mRNA? Here my maths fails me, but intuitively I say that the probability is very very small.
Now, if there are 1,000 different low-abundance mRNAs in the cell the probability of missing each of them is very very small, but the overall probability of missing one of them is 1000 x very very small, so maybe very small.
I'll continue thinking about how to calculate the value of very very small, but if anyone wants to help out - thanks in advance!
What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR?
Polymerase chain reaction (PCR) is a relatively simple and widely used molecular biology technique to amplify and detect DNA and RNA sequences. Compared to traditional methods of DNA cloning and amplification, which can often take days, PCR requires only a few hours. PCR is highly sensitive and requires minimal template for detection and amplification of specific sequences. Basic PCR methods have further advanced from simple DNA and RNA detection. Below, we have provided an overview of the different PCR methods and the reagents we provide at Enzo Life Sciences for your research needs. We aim to help scientists quickly access PCR reagents to use in their next research project!
For standard PCR, all you need is a DNA polymerase, magnesium, nucleotides, primers, the DNA template to be amplified and a thermocycler. The PCR mechanism is as simple as its purpose: 1) double-stranded DNA (dsDNA) is heat denatured, 2) primers align to the single DNA strands and 3) the primers are extended by DNA polymerase, resulting in two copies of the original DNA strand. The denaturation, annealing, and elongation process over a series of temperatures and times is known as one cycle of amplification. Each step of the cycle should be optimized for the template and primer set used. This cycle is repeated approximately 20-40 times and the amplified product can then be analyzed. PCR is widely used to amplify DNA for subsequent experimental use. PCR also has applications in genetic testing or for the detection of pathogenic DNA.
As PCR is a highly sensitive method and very small volumes are required for single reactions, preparation of a master mix for several reactions is recommended. The master mix must be well mixed and then split by the number of reactions, ensuring that each reaction will contain the same amount of enzyme, dNTPs and primers. Many suppliers, such as Enzo Life Sciences, also offer PCR mixes that already contain everything except primers and the DNA template.
Guanine/Cytosine-rich (GC-rich) regions represent a challenge in standard PCR techniques. GC-rich sequences are more stable than sequences with lower GC content. Furthermore, GC-rich sequences tend to form secondary structures, such as hairpin loops. As a result, GC-rich double strands are difficult to completely separate during the denaturation phase. Consequently, DNA polymerase cannot synthesize the new strand without hindrance. A higher denaturation temperature can improve this and adjustments towards a higher annealing temperature and shorter annealing time can prevent unspecific binding of GC-rich primers. Additional reagents can improve the amplification of GC-rich sequences. DMSO, glycerol and betaine help to disrupt the secondary structures that are caused by GC interactions and thereby facilitate separation of the double strands.
RT-PCR & cDNA Synthesis
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a primer complementary to the 3&prime end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
Many RTs are available from commercial suppliers. Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. ProtoScript ® II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
The use of engineered RTs improves the efficiency of full-length product formation, ensuring the copying of the 5&prime end of the mRNA transcript is complete, and enabling the propagation and characterization of a faithful DNA copy of an RNA sequence. The use of the more thermostable RTs, where reactions are performed at higher temperatures, may be helpful for reverse transcription of RNA that containing secondary structure.
For help comparing the RTs and cDNA Synthesis reagents available, view our RT/cDNA Synthesis selection chart.
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Overview of qPCR and RT-qPCR
Quantitative PCR, whether involving a reverse transcription step or not, is routinely used in molecular biology labs and has revolutionized the way in which research is carried out due to its relatively simple pipeline (Figure 2). Its advantages over standard PCR include the ability to visualize which reactions have worked in real time and without the need for an agarose gel. It also allows truly quantitative analysis. One of the most common uses of qPCR is determining the copy number of a DNA sequence of interest. Using absolute quantitation, the user is able to determine the target copy numbers in reference to a standard curve of defined concentration in a far more accurate way than ever before. RT-qPCR, on the other hand, allows the investigation of gene expression changes upon treatment of model systems with inhibitors, stimulants, small interfering RNAs (siRNAs) or knockout models, etc. This technique is also routinely used to detect changes in expression both prior to (as quality control) and after (confirmation of change) RNA-Seq experiments.
Workflow of a standard qPCR and RT-qPCR experiment. Following sample isolation, the integrity is analysed prior to cDNA generation and commencement of the qPCR assay using either intercalating dyes or hydrolysis probes. Fluorescence is detected throughout the PCR cycles and used to generate an amplification curve which is used to quantitate the target sample during data analysis.
The most crucial step in the qPCR and RT-qPCR pipeline is arguably sample isolation. No matter how good your assay design is, if the starting material is contaminated or degraded, you will not get accurate results. A good-quality sample is the starting block of good-quality data. When isolating DNA for qPCR, it is essential that it is free from contaminants that may inhibit the reaction. Most often, extraction is carried out using commercially available kits, which have the advantage of being user-friendly, simple and quick, especially when integrated with a robotic system. The type of RNA extraction carried out depends on the type of RNA required. The most common extraction method used is with total RNA extraction kits. This isolates messenger RNA (mRNA the precursor of protein synthesis), transfer RNA (tRNA decodes mRNA during translation with the ribosome) and ribosomal RNA (rRNA reads the amino acid order during translation and links them with the ribosome), but often (not always) fails to isolate smaller RNAs such as non-coding RNA (ncRNA functional RNA transcribed from DNA, but not translated into proteins) and micro RNAs (miRNAs regulate gene expression by inhibiting mRNA translation). With the explosion of interest in enhancer RNAs (eRNAs small RNAs transcribed from enhancers) which can vary in length considerably, it is essential that the extraction methods are carefully considered to ensure isolation of the RNA of interest. In addition to extraction considerations, it is essential that RNA is not contaminated with DNA, since this cannot be distinguished from cDNA in the qPCR reaction. To overcome this, most protocols rely on the use of a DNase I treatment which digests any DNA.
During isolation, sample degradation is always a possibility. Accordingly, any good pipeline will involve a quality control step to assess the integrity of the sample. This can be done quickly by evaluating the A260/280 ratio (comparing the absorbance at 260 vs 280 nm, a measure of contamination by proteins) and the A260/230 ratio (260 vs 230 nm, an indication of the presence of organic contaminants) of the sample however, this is not very accurate and is subject to interference from several factors. A more accurate measure is the use of a virtual gel electrophoresis system such as the Aligent Bioanalyser. This system works using a chip that separates RNA based on size and detects RNA by fluorescent dyes. This is then translated to a computer which, using an algorithm, produces an RNA integrity number (RIN) which represents the quality of the sample, with 10 being the highest.
The final step in sample preparation for RT-qPCR is the generation of cDNA. cDNA utilizes RT-PCR (Figure 1) to generate cDNA from the RNA template using a reverse transcriptase. This can be done employing oligo(dT) primers, which anneal to the polyA tail of RNA, or using random hexamers (primers of six to nine bases long, which anneal at multiple points along the RNA transcript). Generally, a mix of the two primers is thought to be best as it enables amplification of polyA tail RNA (mainly mRNA) and non-polyA–containing RNA (tRNA, rRNA, etc). In addition to the primer consideration, cDNA generation can be part of the qPCR experiment (termed one-step RT-qPCR) or is generated separately from the qPCR (two-step RT-qPCR), as shown in Figure 3. The advantage of one-step RT-qPCR is that there is less experimental variation and fewer risks of contamination, as well as enabling high-throughput screening hence, this option is usually used for clinical screening. However, it does mean that the sample can only be used a limited number of times, whereas two-step RT-qPCR enables more reactions per sample and flexible priming options and is usually the preferred option for wide-scale gene expression analysis, but does require more optimization.
Human and Mouse Eosinophils Have Antiviral Activity against Parainfluenza Virus
Respiratory viruses cause asthma exacerbations. Because eosinophils are the prominent leukocytes in the airways of 60–70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice after ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726) but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein eosinophil peroxidase, using eosinophil peroxidase–deficient transgenic mice, did not reduce eosinophils’ antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor, Toll-like receptor (TLR) 7. IFNγ increased expression of eosinophil TLR7 and potentiated TLR7-induced nitric oxide production. These results suggest that eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.
Human and mouse eosinophils are antiviral against parainfluenza virus via the production of nitric oxide and by serving as a dead-end host for virus infection, but not through the production of eosinophil granule RNases or eosinophil peroxidase. Eosinophils may have an underappreciated antiviral role in respiratory tract infections in humans.
Eosinophils are found infiltrating the airways of approximately two-thirds of patients with asthma (1), and have been linked experimentally to airway hyperreactivity (2) and airway remodeling (3). Understandably, this has led to the assumption that eosinophils are solely maladaptive in asthma, and, therefore, many asthma treatments are designed to reduce eosinophilic inflammation (4–7). This strategy has produced variable results. Many patients with asthma remain poorly controlled despite maximal therapy (8). Exacerbations of asthma symptoms continue to cause significant morbidity, health care expenditures, and death (9, 10). Moreover, prevention and treatment for the most common cause of asthma exacerbations, respiratory virus infections, are virtually nonexistent (11). These deficiencies highlight our limited understanding of eosinophil biology, and have prompted studies attempting to define the role of eosinophils during respiratory virus infections in asthma. For example, IL-5 transgenic mice with elevated systemic eosinophils had lower titers of respiratory syncytial virus (RSV) compared with wild-type mice (12), whereas IL-5 transgenic mice with airway eosinophilia had lower titers of pneumonia virus in mice compared with controls (13). Similarly, we found that eosinophils recruited to the airways after ovalbumin sensitization were associated with less parainfluenza 1 virus in the lungs of guinea pigs, although those experiments were not designed to establish a causal role for eosinophils (14). Collectively, these studies suggest that eosinophils contribute to antiviral immunity in the lungs.
Eosinophils detect invading viruses via several mechanisms, including Toll-like receptor (TLR)–ligand interactions (15). TLR7 binds single-stranded RNA that is a common genomic motif for many respiratory viruses (16). TLR7 ligation triggers myeloid differentiation primary response 88 (MyD88) adaptor protein–dependent signaling that promotes an eosinophil antiviral response (12). However, the mediators responsible for eosinophils’ antiviral activity are under debate. Eosinophils release a variety of granule proteins after activation that may be antiviral. Eosinophil cationic protein and eosinophil-derived neurotoxin are members of the RNase A superfamily, and are proposed to have a virucidal effect by degrading viral RNA genomes (13, 17, 18). Eosinophil peroxidase is also released from eosinophil granules and contributes to the generation of antimicrobial reactive oxygen species (19). Whether eosinophils target all viruses with these proteins or by unique mechanisms requires further study.
Parainfluenza virus is one of several viruses known to cause asthma exacerbations (20). Parainfluenza is detected in the airways of up to 18% of adults during acute asthma exacerbations (21). Its prevalence is similar to influenza and coronavirus, and significantly greater than RSV in adults. Given that an estimated 16 million acute care visits for asthma exacerbations occur annually (22), parainfluenza-induced exacerbations represent a considerable burden of disease, yet the interaction between parainfluenza virus and eosinophils is not clearly defined.
In this study, we investigated the hypothesis that eosinophils inhibit parainfluenza virus infection in the lungs. The experiments described herein evaluated the effect of eosinophils on parainfluenza virus clearance in vivo and assessed the role of potential antiviral mediators. We also examined the effects of IFNγ on eosinophils’ antiviral responses as a potential mechanism for augmenting eosinophil-mediated antiviral activity.
Mice expressing IL-5 in airway epithelium (NJ.1726) (23), eosinophil-deficient mice (PHIL) (24), and eosinophil peroxidase–deficient mice (25) were maintained by backcrossing on a C57BL/6 background. Animals were handled in accordance with the U.S. Animal Welfare Act. Protocols were approved by Institutional Animal Care and Use Committees of Oregon Health & Science University (Portland, OR) and the Mayo Clinic (Scottsdale, AZ).
Parainfluenza virus (Sendai virus ATCC, Manassas, VA) was grown in rhesus monkey kidney (RMK) cell monolayers, purified by sucrose density centrifugation, and titered in RMK cells (26) (see the online M aterials and M ethods ). Titers were expressed as multiples of the 50% tissue culture infectious dose (TCID50 amount of virus required to infect 50% of RMK monolayers).
Female mice aged 6–8 weeks were sensitized by intraperitoneal injection with 0.04 μg and 0.2 μg ovalbumin (Sigma, St. Louis, MO) with alum on Days 1 and 14, respectively. On Days 24, 26, and 28, mice were anesthetized with ketamine (45 mg/kg) and xylazine (8 mg/kg), and challenged with intratracheal 2% ovalbumin. Mice were infected with parainfluenza (2.8 × 10 4 TCID50 Units) intranasally on Day 27. On Day 31, lungs were lavaged and homogenized (see the online M aterials and M ethods ).
Parainfluenza matrix protein RNA in lung homogenates was amplified by real-time RT-PCR and normalized to 18S. Relative RNA expression was converted to absolute RNA replicates using a parainfluenza RNA standard curve (see the online M aterials and M ethods ).
Eosinophils were isolated to greater than 99% purity and greater than 99% viability from blood of healthy human volunteers by density centrifugation using Ficoll-Paque Plus (Sigma) and negative selection (see the online M aterials and M ethods ). The protocol was approved by the Institutional Review Board. Subjects provided written informed consent.
Human eosinophils were incubated overnight with or without IFNγ (300 U/ml). Medium was removed and parainfluenza (1 × 10 5 TCID50) was added for 2 hours. Some cultures were pretreated with nitric oxide synthase inhibitor, Nω-Nitro-L-arginine methyl ester hydrochloride ([ l -NAME] 100 μM Sigma) 30 minutes before inoculation. Viral infectivity was quantified in RMK cells by hemadsorption assay. Infectious titers are expressed as TCID50 U/ml of culture supernatant.
Human eosinophils were loaded with a nitric oxide–detecting fluorescent probe, (Strem, Newburyport, MA), and exposed to parainfluenza or synthetic TLR7 agonist, see the online M aterials and M ethods . Fluorescence was measured 60 minutes later by plate reader.
Human eosinophils were inoculated with parainfluenza (1 × 10 5 TCID50) for 2 hours, washed to remove unbound virus, and maintained in fresh medium for 72 hours. Viral RNA in culture supernatants was quantified every 24 hours by real-time RT-PCR (see the online M aterials and M ethods ).
Eosinophil TLR7 expression was evaluated by real-time RT-PCR and immunofluorescence (see the online M aterials and M ethods ).
Data were compared using one-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean (±SEM). A P value less than 0.05 was considered significant.
C57BL/6 mice, sensitized and challenged with ovalbumin, had significantly reduced (by ∼90%) parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals ( Figure 1A ). Ovalbumin sensitization and challenge caused a substantial increase in eosinophils in bronchoalveolar lavage fluid, as well as macrophages and neutrophils ( Figure 1B ).
Figure 1. Ovalbumin sensitization and challenge augments clearance of parainfluenza virus infection in the lungs in vivo. (A) C57BL/6 mice were sensitized (intraperitoneal, Days 1 and 14) and challenged (intratracheal, Days 24, 26, and 28) with ovalbumin, and infected with parainfluenza virus (intranasal, Day 27). Parainfluenza virus RNA was quantified by real-time RT-PCR 4 days after infection. Sensitized and challenged mice had significantly reduced parainfluenza RNA in the lung compared with nonsensitized, parainfluenza-infected control mice. (B) Ovalbumin sensitization and challenge increased total inflammatory cells, macrophages (MΦ), eosinophils (Eos), and neutrophils (Neut) in bronchoalveolar lavage fluid compared with nonsensitized controls. There were no differences in lymphocytes (Lymph) between groups (n = 5). *P < 0.05 compared with nonsensitized, parainfluenza-infected control mice. Data are presented as means (±SEM).
NJ.1726 mice (↑Eos ↑IL-5) constitutively express IL-5 in the pulmonary epithelium and have elevated numbers of eosinophils in the lungs (23). At 4 days after infection, parainfluenza RNA was significantly decreased in the lungs of NJ.1726 mice compared with wild-type littermate controls ( Figure 2A ). NJ.1726 IL-5 transgenic mice were crossed with PHIL mice (ØEos), which are congenitally devoid of eosinophils, to differentiate the effect of eosinophils from IL-5. Parainfluenza RNA content in the lungs of IL-5–expressing eosinophil-deficient NJ.1726-PHIL mice (ØEos ↑IL-5) was similar to that in infected wild-type mice, indicating that eosinophils, not IL-5, were mediating the antiviral effect. Similarly, eosinophil-deficient PHIL mice alone had parainfluenza RNA levels comparable to infected wild-type mice ( Figure 2A ). Bronchoalveolar lavage confirmed a significant increase in eosinophils in the airways from NJ.1726 mice compared with wild-type littermate controls, and an absence of eosinophils in PHIL and NJ.1726-PHIL mice ( Figure 2B ). There were no significant differences in the total leukocytes or other inflammatory cell subsets in bronchoalveolar lavage between groups ( Figure 2C ).
Figure 2. Eosinophils promote clearance of parainfluenza virus in the murine lung in vivo. Wild-type (WT) control mice (C57BL/6), transgenic mice with high lung eosinophils due to overexpression of IL-5 by a lung-specific promoter (NJ.1726 ↑Eos ↑IL-5), transgenic mice with high lung IL-5 but devoid of eosinophils (NJ.1726-PHIL ØEos ↑IL-5), and eosinophil-deficient mice (PHIL transgenic mice ØEos) were infected with parainfluenza virus (intranasal). (A) Parainfluenza RNA content in the lung was quantified by real-time RT-PCR 4 days after infection. NJ.1726 mice (↑Eos ↑IL-5) had significantly reduced parainfluenza RNA compared with control mice (WT). NJ.1726-PHIL mice (ØEos ↑IL-5) and PHIL mice (ØEos) had parainfluenza RNA levels comparable to WT. (B) Bronchoalveolar lavage from parainfluenza-infected NJ.1726 mice (↑Eos ↑IL-5) contained significantly more eosinophils compared with all other groups. No eosinophils were detected in the bronchoalveolar lavage in PHIL (ØEos) or NJ.1726-PHIL mice (ØEos ↑IL-5). (C) There were no differences in other inflammatory cell types between groups (n = 7–10). *P < 0.05. Data are presented as means (±SEM).
Eosinophil peroxidase is a granule protein released by eosinophils. Wild-type and transgenic eosinophil peroxidase–deficient mice were sensitized and challenged with ovalbumin, and infected with parainfluenza virus. Parainfluenza virus RNA was quantified from homogenized lung by real-time RT-PCR 4 days after infection. Ovalbumin sensitization and challenge was associated with significantly reduced parainfluenza RNA in both wild-type and eosinophil peroxidase–deficient mice compared with nonsensitized controls ( Figure 3A ), indicating that the eosinophil-mediated antiviral effect was not dependent on the release of eosinophil peroxidase. Sensitized and challenged wild-type and eosinophil peroxidase–deficient mice had significantly elevated airway eosinophils relative to nonsensitized wild-type and eosinophil peroxidase–deficient mice ( Figures 3B and 3C ).
Figure 3. Eosinophil peroxidase is not required for eosinophils’ antiviral effect in vivo. (A) WT mice and eosinophil peroxidase knockout mice (EPX −/− ) were sensitized and challenged (S/C) with ovalbumin and infected with parainfluenza virus. Viral RNA was quantified by real-time RT-PCR 4 days after infection. Sensitized and challenged WT (WT S/C) and EPX −/− mice (EPX −/− S/C) had significantly reduced parainfluenza RNA in the lung compared with nonsensitized, parainfluenza-infected control mice. (B) Ovalbumin sensitization and challenge increased eosinophils in the bronchoalveolar lavage fluid in WT and EPX −/− mice compared with nonsensitized controls. (C) There were no differences in other inflammatory cell types between groups (n = 4–5). *P < 0.05. Data are presented as means (±SEM).
Isolated human eosinophils were inoculated with parainfluenza for 2 hours, then washed to remove unbound virus and cultured in fresh medium for 72 hours to evaluate whether parainfluenza infects eosinophils directly and, if so, whether eosinophils support production of viral progeny. Parainfluenza RNA, quantified by real-time RT-PCR from culture supernatants, progressively increased over 72 hours ( Figure 4 , left axis), indicating that the virus is capable of infecting eosinophils and producing viral transcripts. However, despite replication, the parainfluenza virus titers, as determined by hemadsorption assay, remained undetectable at these time points ( Figure 4 , right axis), indicating that eosinophils do not support production of infectious virus.
Figure 4. Parainfluenza-infected eosinophils produce viral progeny that are not infectious. Human eosinophils were isolated from the peripheral blood and infected with parainfluenza virus for 2 hours, washed to remove unbound virus, and maintained in culture for 72 hours. Parainfluenza RNA from eosinophil supernatants was quantified by real-time RT-PCR every 24 hours (solid square, left axis). Concurrently, infectious parainfluenza virus titers were assessed by hemadsorption assay using rhesus monkey kidney cells and expressed as 50% tissue culture infectious dose (TCID50) U/ml (open circle, right axis) (n = 4). Data are presented as means (±SEM).
The levels of RNA encoding three parainfluenza virus structural proteins (i.e., matrix protein, fusion protein, and nucleoprotein) were compared 2 hours after inoculation between virus-infected eosinophil cultures and cultures with virus-inoculated media to determine if the eosinophil-mediated antiviral effect resulted from the release of eosinophil granule RNases. Concurrently, parainfluenza virus titers were assessed at this time point in RMK cells by hemadsorption assay. Eosinophils significantly decreased parainfluenza virus titers compared with virus-inoculated cultures without eosinophils, showing that eosinophils reduce parainfluenza virus infectivity ( Figure 5A ). A further reduction in virus titers was seen when eosinophils were pretreated with IFNγ. However, the quantities of RNA for all three parainfluenza virus structural proteins were similar between cultures containing virus-infected eosinophils and virus-inoculated cultures devoid of eosinophils ( Figure 5B ), demonstrating that reduced infectivity was not due to the degradation of the viral RNA genome by the eosinophil granule RNases.
Figure 5. Eosinophils’ antiviral activity against parainfluenza does not involve granule RNases. Human eosinophils were isolated from peripheral blood and infected with parainfluenza virus. (A) Viral infectivity was assessed 2 hours after inoculation by hemadsorption assay and expressed as viral TCID50 U/ml. Parainfluenza infectivity was nearly undetectable in the supernatants of cultured eosinophils (Eos) and eosinophils pretreated with IFNγ (Eos + IFNγ) compared with parainfluenza virus cultured in media without eosinophils (Media + IFNγ). (B) RNA encoding parainfluenza virus structural proteins, matrix, fusion, and nucleoprotein (NP) was quantified in eosinophil supernatants by real-time RT-PCR 2 hours after inoculation. No differences were detected between any of the three genes, suggesting that the reduction in parainfluenza infectivity was not due to viral RNA degradation by eosinophil granule RNases (n = 4–7). *P < 0.05. Data are presented as means (±SEM).
Parainfluenza virus was incubated in the presence of isolated human eosinophils for 2 hours and then viral infectivity was quantified by titering in RMK cells (via hemadsorption assay). As shown in Figure 6A , eosinophils significantly decreased parainfluenza virus titers compared with parainfluenza incubated in media without eosinophils. Eosinophils were cultured with the nitric oxide synthase inhibitor, l -NAME, to determine if the eosinophil-mediated antiviral effect was due to nitric oxide production. Eosinophils treated with l -NAME lost their ability to reduce virus titers, indicating that, upon exposure to virus, eosinophils attenuate viral infectivity through the production of nitric oxide ( Figure 6A ). The quantity of nitric oxide produced by eosinophils was evaluated with a copper-conjugated intracellular nitric oxide–detecting fluorescent probe 1 hour after addition of virus. Eosinophils exposed to parainfluenza virus had significantly increased fluorescence, indicating that eosinophils produce nitric oxide in response to parainfluenza infection. This increased fluorescence was blocked by l -NAME, confirming that nitric oxide was the mediator responsible ( Figures 6B and 6C ).
Figure 6. Eosinophil-derived nitric oxide reduces parainfluenza virus infectivity. Human eosinophils isolated from peripheral blood were cultured overnight with IFNγ. (A) Eosinophils were treated with the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride ( l -NAME 100 μM) or vehicle for 30 minutes, then infected with parainfluenza virus. Virus infectivity was quantified 2 hours after infection by hemadsorption assay and expressed as viral TCID50 U/ml. Parainfluenza virus infectivity was significantly decreased in the presence of eosinophils. Eosinophils treated with l -NAME lost their antiviral activity (n = 4). (B) Nitric oxide production was assessed using an intracellular nitric oxide–detecting fluorescent probe. Eosinophils loaded with the probe were treated with l -NAME or vehicle and infected with parainfluenza for 1 hour. Eosinophil fluorescence increased significantly in response to parainfluenza infection, indicating an increase in nitric oxide production by eosinophils. l -NAME treatment prevented parainfluenza-induced nitric oxide production by eosinophils (n = 4). (C) Representative images of eosinophils loaded with nitric oxide–detecting fluorophore. Magnification, ×100. *P < 0.05. Data are presented as means (±SEM).
Eosinophils were cultured with or without IFNγ overnight. IFNγ increased eosinophil TLR7 expression relative to unstimulated eosinophils, both quantitatively by real-time RT-PCR ( Figure 7A ) and qualitatively by immunostaining ( Figure 7B ). Eosinophils’ response to TLR7 agonist was also evaluated with a nitric oxide–detecting fluorescent probe. Eosinophils treated for 1 hour with the synthetic TLR7 agonist, R837, had significantly increased fluorescence compared with vehicle-treated controls, indicating that TLR7 agonist induces nitric oxide production ( Figures 7C and 7D ). R837-mediated nitric oxide production was potentiated in eosinophils exposed to IFNγ. l -NAME and the oligonucleotide TLR7 antagonist, IRS661, but not a control oligonucleotide, blocked R837-induced nitric oxide production.
Figure 7. IFNγ up-regulates Toll-like receptor 7 (TLR7) expression and potentiates TLR7-induced nitric oxide production in eosinophils. Human eosinophils were isolated from peripheral blood and grown in culture. (A) TLR7 expression was quantified by real-time RT-PCR from eosinophils treated with or without IFNγ. TLR7 RNA was significantly increased in eosinophils treated with IFNγ compared with untreated eosinophils (n = 5). (B) Eosinophils were immunostained with antibodies against TLR7 (green), and nuclei were labeled with 4′,6-diamidino-2-phenylindole (blue). TLR7 was expressed in unstimulated (−IFNγ) and IFNγ stimulated (+IFNγ) eosinophils. Images obtained with an LSM780 confocal microscope (numerical aperture 1.4 Zeiss, Thornwood, NY). (C) Eosinophils were loaded with an intracellular nitric oxide–detecting fluorescent probe and stimulated with synthetic TLR7 agonist R837. Eosinophil fluorescence increased in response to R837. IFNγ pretreatment potentiated the R837-induced increase in fluorescence. R837-induced fluorescence was blocked by l -NAME and by the oligonucleotide TLR7 antagonist IRS661, but not by a control oligonucleotide (IRS control) (n = 6). (D) Representative images of eosinophils loaded with nitric oxide–detecting fluorophore. Magnification, ×100. *P < 0.05 compared with no IFNγ control. # P < 0.05 compared with all other groups, except between IFNγ-treated R837 and R837 + IRS control. Data are presented as means (±SEM).
Our results demonstrate a significant antiviral effect of eosinophils on parainfluenza virus infection in mouse airways in vivo and in isolated human eosinophils in vitro. Eosinophils appear to mediate their antiviral effects via both passive and proactive mechanisms. Specifically, our data show that eosinophils are a “dead-end” cellular host for parainfluenza, failing to propagate infectious viral progeny after infection. Thus, elevated eosinophil numbers in the lung may passively disrupt airway infection by acting as a cellular decoy and/or “sink,” interfering with the progressive expansion of viral numbers during infection. Our data also show that eosinophils proactively mediated antiviral activities through the production of nitric oxide and up-regulation of TLR7. In contrast, we did not find evidence that eosinophils directly attenuate parainfluenza through the release of eosinophil peroxidase or by degrading the viral RNA genome through the release of granule RNases.
These findings expand on prior eosinophil studies (12–14, 17, 18, 27) by establishing an antiviral role for eosinophils against parainfluenza virus, while suggesting that key differences exist in the eosinophils’ antiviral mechanisms against different viruses. Previous studies proposed that eosinophils inhibit RSV and pneumonia virus in mice, in part via the degradation of viral RNA genomes by eosinophils’ granule RNases, eosinophil cationic protein, and eosinophil-derived neurotoxin (13, 17, 18). However, we found that eosinophils did not alter the quantity of viral RNA transcripts for three key parainfluenza virus structural proteins (i.e., matrix, fusion, and nucleoprotein), despite causing a substantial reduction in parainfluenza infectivity, titered in RMK cells by hemadsorption assay. This suggests that eosinophils do not inhibit parainfluenza through RNase activity on the viral genome. Importantly, our methods differed from prior studies by quantifying both viral RNA and infectivity compared with only assessing viral infectivity after treating infected eosinophils with RNase inhibitors. Concurrent measurement of viral RNA levels and infectivity led to the additional finding that, although human eosinophils infected with parainfluenza produce viral RNA, these viral progeny are not infectious. This phenomenon, termed an “abortive” infection, has been described for many viruses, including RSV (28) and coronavirus (29) in macrophages, and influenza A in neutrophils (30), as well as for parainfluenza virus in Madin-Darby bovine kidney cells (31) and chick embryo fibroblasts (32). Evidence suggests that specific virus and host cell characteristics impact productive versus abortive outcomes during virus infections (33), but those characteristics require further investigation.
Eosinophils also inhibit parainfluenza through the production of nitric oxide. Previously, systemic suppression of nitric oxide production using an inhibitor of nitric oxide synthase delayed clearance of RSV in wild-type mice and in IL-5 transgenic animals with systemic eosinophilia in vivo (12, 34). Given the widespread expression of nitric oxide synthases, however, these studies were unable to distinguish the specific role of eosinophil-derived nitric oxide, which our study has elucidated. The ability of eosinophils to produce nitric oxide has been suspected for some time, given the presence of both constitutive and inducible nitric oxide synthase isoforms in eosinophils (35), but detecting nitric oxide has historically been challenging due to its short half-life and rapid diffusion across biologic membranes (36). These issues were overcome by using a copper-conjugated intracellularly retained fluorescent probe. We found that eosinophils generate nitric oxide in response to virus infection, and in response to stimulation with synthetic TLR7 agonist. IFNγ potentiated TLR7-mediated nitric oxide production in eosinophils, likely in part through the up-regulation of TLR7 expression. Thus, our results show that antiviral cytokine signaling augments eosinophils’ viral detection and their antiviral nitric oxide response.
Nitric oxide mediates antiviral effects via the production of a variety of reactive nitrogen products that inhibit viral proteases (37), and alter host cell function through nitrosylation of tyrosine and thiol groups (38). The consequences of these nitrogen species are diverse, ranging from inhibition of virus protein and RNA synthesis to potentiation of inflammation and injury to the respiratory tract (39). Nitric oxide also regulates many other physiologic processes, including eosinophil migration (40) and survival (41, 42), airway epithelial cell ciliary motility (43), and airway caliber through airway smooth muscle relaxation (44). Eosinophil-derived nitric oxide may affect multiple functions in the airway beyond what our experiments were designed to assess.
Eosinophils’ attenuation of parainfluenza virus infection was not dependent on the release of eosinophil peroxidase. Eosinophil peroxidase is a granule protein that, together with the respiratory burst from activated eosinophils, generates potent reactive oxygen species that have been suggested to contribute to host immune defense (45). For example, eosinophil peroxidase has been shown to have virucidal effects against human immunodeficiency virus in vitro (46). However, the role of eosinophil peroxidase in vivo as a host defense mechanism remains unclear. Eosinophil peroxidase deficiency had no effect in an experimental model of helminthic parasite infections in mice (47), and its deficiency has also been detected in humans without manifesting an apparent phenotype (48). Our data further suggest that eosinophil peroxidase has no role in eosinophils’ antiviral activity against parainfluenza.
Importantly, our experiments differentiate the effects of eosinophils’ versus IL-5. Although NJ.1726 IL-5 transgenic mice (↑Eos ↑IL-5) with an abundance of airway eosinophils had reduced parainfluenza virus in the lung, NJ.1726-PHIL mice (ØEos ↑IL-5) with elevated IL-5 in the absence of eosinophils did not. This is pertinent given the presence of IL-5 receptors on leukocytes other than eosinophils, including macrophages and neutrophils (49), which could have contributed to the antiviral effect in vivo. Furthermore, our data demonstrate that, although an induced airway eosinophilia promoted viral clearance, clearance of virus was similar for eosinophil-deficient mice relative to wild-type control animals. This suggests that the presence of a specific airway eosinophilia mediates one or more unique antiviral mechanisms not occurring at homeostatic baseline, and underlines the need for better characterization of asthma phenotypes when evaluating respiratory virus infections in humans.
Human studies have historically been confounded by the heterogeneity of asthma phenotypes (50), the need for invasive bronchoscopic testing, the immune effects of corticosteroid treatment, differences in virus detection methods (i.e., PCR, ELISA, culture) and the variety of respiratory viruses that cause asthma exacerbations (20). Recent trials that studied exacerbation rates in well characterized steroid-resistant eosinophilic subjects with asthma treated with mepolizumab, a monoclonal antibody against IL-5, found no difference in the incidence of respiratory tract infections (4, 5). However, airway infections were rare events, and were diagnosed clinically without objective evidence of the presence of an infecting virus. Furthermore, these trials did not quantify airway and lung parenchymal eosinophils, nor did they characterize the eosinophils’ activation state after treatment. Nonetheless, future studies that define the interaction between eosinophils and viruses may lead to novel treatment targets for eosinophilic asthma and for respiratory virus infections in humans.
The results of our study paradoxically suggest that eosinophils have beneficial antiviral effects during respiratory virus infections despite considerable evidence linking viruses to asthma exacerbations (20, 21). However, we cannot conclude that eosinophils’ antiviral effects result in fewer exacerbations. On the contrary, it is possible, and perhaps likely, that eosinophils’ ability to detect and respond to viruses promotes an exuberant, and ultimately detrimental, airway inflammatory response in subjects with asthma. Although our study did not specifically address airway physiology, Adamko and colleagues (14) found that eosinophils mediate virus-induced airway hyperreactivity in sensitized guinea pigs, and were associated with lower viral titers, yet lower viral titers did not improve airway hyperreactivity. Thus, the negative effects of activated eosinophils’ in the airway may mask any positive impact from fewer viral transcripts. Consequently, as therapies become progressively more targeted against pulmonary eosinophils, there will be an even greater need for understanding the complexity of eosinophil biology in the lungs.
Advantages & Limitations of scRNA-Seq Assays
scRNA-Seq is a very powerful method that allows transcriptomic analysis of heterogeneous tissue, or dynamic processes in one single experiment. Without microdissection or FACS-sorting, samples can be directly prepared for the scRNA-Seq protocol. Depending on the number of cells and the sequencing depth, scRNA-Seq can be very sensitive and detect a population representing less than 1% of the total population.
Because of the quantity of information generated by scRNA-Seq, some of the protocol steps have to be handled with care. The preparation of the single-cell solution can be challenging especially for tissues or cells surrounded by an extracellular matrix. The protocols used to isolate these cells can by themselves modify the transcriptome and the sequencing data could be the result of these manipulations instead of an endogenous cellular process. In contrast, for bulk RNA-Seq, tissues can be directly lysed in trizol, freezing the transcriptome at the very first step of the extraction. scRNA-Seq identifies fewer transcripts than bulk RNA-Seq and this imperfect coverage can lead to a biased quantification. However, this flaw can be corrected by bioinformatic analysis. Finally, most scRNA-Seq protocols only focus on poly-A RNAs, which excludes all non-coding RNAs.
scRNA-Seq is more expensive and more time-consuming than bulk RNA-Seq but in one experiment, you obtain the transcriptome profile of several populations.
Pulmonary Adenocarcinoma—Pathology and Molecular Testing
Prodipto Pal MD, PhD , . Ming-Sound Tsao MD, FRCPC , in Pulmonary Adenocarcinoma: Approaches to Treatment , 2019
Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) is feasible in clinical laboratories, however, with its own set of challenges. As noted earlier, ALK rearrangement has many different candidate fusion partners, even for the ALK-EML4, the most common fusion in NSCLC, there are many fusion variants, largely due to different breakpoint regions on EML4, thus would require a multiplexed approach. However, with RT-PCR-based methods, a priori knowledge of the candidate fusion genes is needed to identify fusion variants which can later be confirmed by subsequent sequencing 101 and has been used in studies with high level of sensitivity albeit with varying level of specificity (85%–100%) compared with FISH. 102,103 A negative result by RT-PCR requires appropriate caution due to the potential of false-negative results due to the missing unknown fusion variants. In addition, RT-PCR assays are dependent on procuring high-quality RNA from FFPE tissue. Newer RNA-based assays are under active development (assays such as NanoString system) that can simultaneously detect various fusion partners as well as offer the added advantage of detecting other driver fusion-type alterations in a multiplexed setup (such as ROS1, RET, etc.). 104–106
In summary, we described an alternative method using Real-Time RT-PCR to determine the rate of mRNA degradation by accurately measuring the number of mRNA molecules relative to total RNA. Using this approach, we obtained a values for the β-actin mRNA half-life in Nalm-6 cells that are comparable to those estimated from Northern blot studies using normal human leukocytes . Therefore, Real-Time RT-PCR is a reliable method for measuring mRNA half-life. Because of its sensitivity, half-life of mRNAs expressed at very low level can be determined in cases in which Northern blots may not be sensitive enough. The length of the amplified fragment is important to determine the mRNA half-life because incomplete or degraded mRNA can interfere with the measurement of the actual mRNA half-life. Ideally, full-length cDNA molecules should be amplified to ensure integrity and identity of the mRNA species. The use of the fluorogenic SYBR Green I dye limits the length of the amplified product (cDNA recommended to be less than 200 bp). However, the use of TaqMan ® (Applied Biosystems, Foster City, CA, USA), molecular beacons (Molecular Probes, Inc., Eugene, OR, USA), or fluorescence resonance energy transfer (FRET) probes (Roche Molecular Biochemicals, Indianapolis, IN, USA) could overcome this limitation and allow amplification of longer PCR products. In addition, since multiplex Real-Time RT-PCR can be achieved in the same reaction tube using different fluorogenic dyes, this method could be modified to simultaneously estimate mRNA half-life of several genes. Thus, our approach represents a rapid and sensitive assay to determine mRNA half-life.
Sars-CoV-2 RNA on surfaces poor indicator of quantity, timing of previous contamination
Novel Coronavirus SARS-CoV-2 Transmission electron micrograph of SARS-CoV-2 virus particles, isolated from a patient. Image captured and color-enhanced at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: National Institute of Allergy and Infectious Diseases, NIH
A team of UK investigators has shown that RNA copies recovered from surfaces are a poor indicator for determining the numbers of viable SARS-CoV-2 virus particles.
The research, published in Applied and Environmental Microbiology, a publication of the American Society for Microbiology, found that a common UK isolate of SARS-CoV-2 that may have initially contaminated surfaces in the general environment became undetectable within 48 hours. The isolate of SARS-CoV-2 remained viable for more than twice as long on hydrophobic surfaces as on hydrophilic surfaces.
The materials tested in the study were representative of those in personal protective equipment, as well as high hand-touch sites such as stainless steel.
An impediment to determining how long SARS-CoV-2 remains viable on different surfaces has been that in most cases, viable virus that has landed on surfaces is rarely detected, according to corresponding author Thomas Pottage, BSc, Senior Project Leader—Biosafety and Bioresponse, Public Health England, Porton Down, UK. However, many studies have used reverse transcription polymerase chain reaction (RT-PCR) successfully to detect viral RNA and use this as an indicator of how much of the virus was previously found on the surface.
Therefore, the investigators first sought to determine the relationship between initial quantities of viable virus, and subsequent RNA copy number on a contaminated surface, hoping that the latter could serve as a surrogate for the former. To test this, they contaminated surfaces of interest with artificially high concentrations of the virus in the laboratory. But there was no clear relationship between the numbers of RNA copies recovered over time to the number of viable virus particles in the initial inoculum. The investigators did observe that the numbers of virus particles decreased quite rapidly.
The relationship in levels of RNA and viable SARS-CoV-2 shows that in previous surface sampling studies where SARS-CoV-2 RNA was recovered, there were likely low concentrations of viable virus on those surfaces at the time of contamination, according to Pottage.
"This study estimates that the SARS-CoV-2 would survive less than two days on surfaces under normal environmental concentrations," said Pottage.
Conversely, artificially high concentrations of SARS-CoV-2 that were deliberately deposited on surgical mask material and stainless steel in a laboratory setting resulted in relatively lengthy persistence, with 99.9% reductions in viable virus taking place over 122 and 114 hours, respectively. Viability dropped the fastest on a polyester shirt, with a 99.9% reduction occurring over 2.5 hours.
"Our results show that the porous but hydrophobic surfaces of the surgical mask and Tyvek coverall produce similar decay rates when compared to the non-porous hydrophobic surfaces of stainless steel," with a reduction in numbers of virus particles of 99.999% over seven days, said the authors. Viable SARS-CoV-2 was recovered from these surface materials over longer periods of time compared to the porous, hydrophilic surfaces tested, cotton and woven polyester.
Small RNA Detection by in Situ Hybridization Methods
Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.
Keywords: LNA probe PLA Piwi-interacting RNA TIRCA enzyme-labeled fluorescence signal amplification padlock probes rolling circle amplification short interfering RNA tyramide signal amplification.
Types of nucleotide modifications used…
Types of nucleotide modifications used in small RNA ISH probes. ( A )…
Sequence amplification and detection methods…
Sequence amplification and detection methods for small RNA ISH include ( A )…
Sequence amplification and detection methods…
Sequence amplification and detection methods for small RNA ISH include ( A )…