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FAQs

  • Miscellaneous Questions
    • What is the purpose of the 25C incubation step when using Q-Script-RT during cDNA synthesis?
      The purpose of the 25C incubation step during cDNA synthesis is to allow annealing and extension of the random primers. Since the primers are short, a lower temperature is required. Omission of this step will result in inefficient cDNA synthesis.
    • How are PerfeCTa® microRNA assays designed?
      PerfeCTa microRNA assays are designed with primer design software according to the following criteria: • Optimized primer Tm designed to match the Universal PCR Primer • Universal cycling conditions to ensure robust amplification for all assays in profiling experiments • No self-complementarity or primer dimer artifacts with the PerfeCTa Universal PCR Primer • Optimized PCR product size with melting temperature of 75-78°C
    • Do the PerfeCTa microRNA assays distinguish between closely related family members?
      PerfeCTa microRNA Assays will distinguish closely related family members are distinguished to varying degrees depending on the specific assay. The current microRNA assays have been designed primarily as a general profiling reagent that work with maximum efficiency with common PCR cycling conditions. Greater assay specificity can be achieved by increasing the annealing temperature of the PCR cycling from 60°C to 63°C with some cost to assay sensitivity. In some cases assays have been designed (for example, the let-7 family) that will distinguish closely related family members.
    • How are PerfeCTa microRNA assays validated?
      • Proper tissue or cell type specificity (where applicable) • No primer dimer or off-target amplification product • Good amplification efficiency tested with multiple input amounts of cDNA • Comparison of qPCR results to a no-poly(A) polymerase control must demonstrate significant differences in samples where the microRNA is present • A single melt peak observed at the expected amplicon melt temperature
    • What should I do if I suspect that my RNA contains RNase activity?
      If it is suspected that an RNA prep contains RNase activity, add RNase inhibitor to the reverse transcription reaction at a final concentration of 0.4 U/µl. Q-Script RT is a mixture of MMLV RT reverse transcriptase and Rnase inhibitor and should provide some safeguard against this problem. Q-Script RT is used in all of Quanta's cDNA synthesis kits: qScript™ cDNA Synthesis Kit (Cat# 95047), qScript™ cDNA SuperMix (Cat# 95048), qScript™ Flex cDNA Kit (Cat# 95049). If the RNA is grossly contaminated, another RNA prep should be used.
    • What are the sources of non-specific amplification products?
      The origin non-specific amplification could potentially arise from mRNA transcripts containing sequences similar to the microRNA assay sequence close to their 3’-ends. Specificity of the microRNA assay comes from a single microRNA-specific assay primer, thus, there is a slightly higher probability of non-specific amplification for microRNA detection than a typical two-step RT-qPCR assay for mRNA which employs two gene-specific primers. In general, amplification products are not produced from single primer reactions (microRNA-specific primer alone or UAP alone). Positive signals are dependent on inclusion of both the UAP and the microRNA-specific primer in the qPCR. The probability non-specific amplification products increases with increasing cDNA template in the qPCR reaction and when the microRNA of interest is rare or absent from the RNA sample. When observed, non-specific amplification can be reduced by increasing the temperature of the reverse transcriptase reaction to 45°C without compromising assay sensitivity. In addition, increasing the annealing temperature of the PCR cycling condition (up to 63°C) will reduce non-specific amplification at some cost to assay sensitivity. The most useful control to measure non-specific amplification is the no-poly(A) polymerase (no-PAP) control. Assay results should be considered negative if the difference in CTs from the plus-PAP and no-PAP reactions is less than 2 CTs.
    • What is the optimal amount of RNA input?
      The kit can is quantitative with 1 ug to 10 pg of total RNA in a 20 uL cDNA reaction. More than 1 ug can be used in larger reaction volumes scaled appropriately.
    • Does the RNA need to be DNAse treated?
      DNAse treatment is not necessary and not recommended for microRNA detection. qPCR reactions of no-RT controls or using genomic DNA as template do not produce amplification products. DNAse is often difficult to inactivate and residual DNAse activity will greatly decrease sensitivity of any RT-qPCR assay.
    • What assays should I use for normalization of the data?
      Use any assay or set of assays that are stable (do not change) with respect to the treatment or conditions of your experiment in you biological model system (for example control vs. treated or normal vs. disease). You can learn much more about the subject at: http://normalisation.gene-quantification.info/. Click on microRNA on the panel on the left and click on microRNA normalization link at the top of the microRNA page.
    • Why are the PAP and RT reactions not combined into a single reaction?
      A combined poly(A)-tailing and reverse transcription reactions means compromising optimal assay conditions for both steps and reduces the flexibility of the system to accommodate different end user applications. For example, the PAP reaction can be adjusted and scaled to accommodate a wide range of RNA inputs. When using less than 100 ng of total RNA the PAP reaction can be shortened to 20 minutes and/or less PAP enzyme can be used in the reaction. When using more than 1 ug of total RNA the PAP reaction can be scaled up and stored for later use into multiple RT reactions. The specificity of some microRNA assays can be increased by increasing the temperature of the cDNA synthesis reaction from 42 ˚C to 45 ˚C or higher which is good for RT but not so good for PAP. Combining the two reactions does not permit a no-poly(A) polymerase control which is a critical measurement of microRNA assay background signal and allows for detection of false-positive signals. In addition, the poly(A) polymerase would have the potential to tail the oligo dT adapter primer interfering with specificity of the cDNA synthesis reaction.
    • What are the -3p and -5p designations on each microRNA?
      Previously the mature microRNAs were referred to as major miRs and minor miRs (according to their relative abundance in specific tissues) with the minor miRs being designated with an asterisk. The nomenclature at miRBase has now changed such that for each precursor microRNA there are potentially two mature microRNAs designated with a -5p and -3p which refer to the position (5-prime arm or 3-prime arm) that the mature microRNAs occupy within the precursor stem-loop structure. On the PerfeCta microRNA Assay website click on the links for both the -5p and -3p microRNAs. In red text there is a reference to the former miRBase IDs. There is also a link to the miRBase Entry (blue button) where you can cross-check all of the information.
    • What should I do if I see a split cluster on either axis?
      This usually occurs if an alternate SNP site is present in the template, in the region complementary to the SNP-IT primer. This potential single base mismatch in some of the samples, at the alternate SNP site, may cause inefficient SNP-IT primer/template hybridization. Even though the extension step occurs, the signal is weaker in these samples and hence will show up as a distinct cluster in the scatter plot. This can be overcome by redesigning the SNP-IT for the opposite strand and on the other side of the SNP, so that the alternate SNP site is avoided altogether at the SNP-IT annealing step.
    • Should I be concerned if I observe three clusters in the scatter plot but one of them seems to be shifting to the other or are very close to each other?
      The observation is usually due to template dependent noise. This happens when the SNP-IT primer anneals to more than one site on the PCR template, other than its intended location (immediately adjacent to the SNP of interest). This phenomenon leads to multiple extensions occurring at different sites and often produces a significant level of background in the SNP-IT assay. This is commonly observed as a shift towards heterozygotes in one or more of the genotype clusters. Since this is template specific, it is usually best to choose an alternate design for the assay primers.
    • What should I do if my homozygous samples controls look like heterozygotes in the assay?
      The observation is usually due to template dependent noise. This happens when the SNP-IT primer anneals to more than one site on the PCR template, other than its intended location (immediately adjacent to the SNP of interest). This phenomenon leads to multiple extensions occurring at different sites and often produces a significant level of background in the SNP-IT assay. This is commonly observed as a shift towards heterozygotes in one or more of the genotype clusters. Since this is template specific, it is usually best to choose an alternate design for the assay primers.
    • How much of the first strand reaction should I add to the PCR?
      The volume will depend on the starting amount of RNA used for first-strand synthesis, and the abundance of the target gene. We recommend starting with 10% of the first-strand reaction. More than 10% may inhibit the PCR.
    • How do I eliminate non-specific bands in PCR?
      Here are some suggestions for optimizing your PCR under such conditions: - Make sure primers don't have complementary sequences at the 3' ends - Optimize the annealing step by increasing the temperature in 2-5C increments, and minimizing the annealing time. You can try higher annealing temperatures in the first few cycles, and lower annealing temperatures in the subsequent cycles. - Try hot-start protocols - Optimize the magnesium concentration for each template and primer combination - To minimize chances of amplifying contaminating DNA, use aerosol-resistant tips and UDG
    • What is the control date? (expiration Date)
      The control date is not the expiration date, but rather the date through which we guarantee performance of the product. If stored under the recommended conditions, the product will maintain performance through the date printed on the label.
    • One-step versus Two-step RT-PCR
      One-Step RT-PCR allows easier processing of large numbers of samples, and helps minimize carry-over contamination since tubes are not opened between cDNA synthesis and amplification. By amplifying the entire cDNA sample, one-step RT-PCR can provide greater sensitivity-down to 0.01 pg total RNA. You can only use gene specific primers with these kits. Two-Step RT-PCR is useful for detecting multiple messages from a single RNA sample. You’ll get greater flexibility when choosing polymerase and primers than with one-step RT-PCR systems. When performing two-step RT-PCR you have the option of using either oligo(dT), random hexamers, or gene-specific primers, and then performing PCR in combination with either AccuStart Taq DNA Polymerase, or your choice of other PCR enzymes.
    • What controls should be run, no-PAP, no-RT or both and why?
      When the Ct values are high (approaching 30) a no-PAP control can be used to add confidence in the results. If there is a significant difference between the plus-PAP and no-PAP reactions the results can be considered real. The no-RT control should always be negative. The kit comes with 20% extra 5x PAP reaction buffer and cDNA mix to accommodate the use of controls.
    • Is the use of UNG necessary for performing reverse transcription reactions?
      No. It is not recommended to use UNG when performing reverse transcription. When using a dNTP mix with dUTP in a RT reaction, uracil will be incorporated into the cDNA generated from your RNA template. UNG (uracil N-glycosylase) is capable of cleaving single- or double stranded DNA containing dUTP sequences. Therefore, use of UNG during a reverse transcription step will cleave the dU containing cDNA and result in significantly lower amplification or absence of amplification.
    • I am using Uracil N-glycosylase (UNG) and dUTP in my PCR reactions and would like to use the PCR product in a post-PCR application. Does the dUTP affect my ability to hybridize, sequence, clone, or digest the PCR product?
      Uracyl residues are roughly equivalent to dT-containing PCR products as hybridization targets, if long fragments (>200bases) are used. With very short fragments (<30bases), hybridization of dU containing templates will require lower temperatures depending on the dU content of DNA. Uracil residues serve in an equivalent manner as dT-containing PCR products as templates for dideoxy-terminated sequencing reactions. Uracyl residues are equivalent to dT-containing PCR products if transferred into UNG-minus bacterial hosts as targets for direct cloning. The recognition of dU-containing DNA by restriction endonucleases has been studied. Depending on the specific endonuclease, there may be no effect of the substitution of dU for dT on enzymatic activity (e.g., EcoR1 and BamH1), or the dU-containing DNA is cleaved more slowly than dT-containing DNA (e.g., Hpa1, HindII, and HindIII). For other endonucleases the effect of substituting dU for dT on enzyme activity will need to be examined empirically on an individual enzyme basis.
    • What is UNG (Uracil N-glycosylase)?
      UNG (Uracil N-glycosylase) is an enzyme used in a powerful method for the elimination of carryover PCR product. This method modifies the PCR products such that the products from previous PCR amplifications will be digested by UNG prior to initiation of amplification. During amplification dUTP is substituted for dTTP resulting in dUTP containing products. UNG is active on single and double stranded dUTP containing DNA. A short pre-PCR incubation step in subsequent reactions will allow the UNG to digest any dUTP containing DNA. Since UNG is active on single and double stranded dUTP containing DNA, the procedure should work on dU-containing PCR products from standard or asymmetric PCR amplifications. Uracil ribonucleotide residues in RNA, novel DNA containing dTTP or cDNA containing dTTP are not suitable substrates for UNG. This method is best put in place prior to the appearance of contamination problem, because it is effective only against contamination with dUTP labeled PCR products.
    • Purpose of the 25C incubation step when using Q-Script RT and Rnase Inhibitor mix
      The purpose of the 25 C incubation step when using random hexamers is to allow annealing and extention of random primers. Since the primers are short, a lower temperature is required. Omission of this step will result in inefficient cDNA synthesis.
    • Inactivation of reverse transcriptases: protocol.
      The enzymes can be inactivated by adding a chelating agent such as EDTA. They should be heated to 85°C for 5 minutes for complete inactivation.
    • M-MLV RT: Storage & Stability
      MMLV RT in the Q-Script RT and Rnase Inhibitor mix is stable up to 2 years when stored at -20°C in a non-frost-free freezer. Enzymes may remain at 4°C for up to 48 hours without loss of activity
    • What is the highest temperature that a reverse trascriptase can be used?
      The optimal temperature for MMLV is 42 C. Therefore, for optimal results, we recommend carrying out cDNA synthesis reactions at 42°C. Only in rare cases, such as One-Step qRT-PCR, where shorter and more specific RNA regions are transcribed, may it be effective to raise the temperature to 48° although a slight reduction in RT activity and half-life may occur at these temperatures. Discuss 1 step Kit temp. 45-50 and 2step kit protocols
    • Mw and Size of Taq Polymerase
      Taq DNA Polymerase is an 832 amino acid, single subunit enzyme with a MW of 94,000.
    • Optimal pH for Taq polymerase
      Taq polymerase is active from pH 7.5-9.5. The unit assay is performed at pH 9.3 in TAPs buffer.
    • Extension rate of Taq
      Taq has been reported to have an extension rate of 35-100 nt/sec at 75°C. For further information, see Wittwer (1991) BioTechniques 10(1), 76. It should be noted that the extention rate will vary depending on the conditions in which the enzyme is being used.
    • Is a probe assay more sensitive than a SYBR® Green I assay?
      A probe assay and a SYBR® Green I assay can be equally sensitive. In cases of difficult to optimize PCRs the SYBR® Green I assay might be less beneficial as it shows the total fluorescent signal of primer dimers, aspecific product and wanted product.
    • What is the advantage of working with a probe system?
      A probe system is always specific (except Amplifluor™ probes) and therefore does only detect the gene of interest. If you have a difficult to optimize PCR it will not show you any primer dimers or aspecific products. With a probe system it is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimisation than SYBR® Green I assays.
    • What is the advantage of working with SYBR® Green I?
      SYBR® Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in Real-Time. This dye is mainly very attractive for researchers analysis lots of different genes. However it is important to do a good primer design to avoid primer dimers, which will also be detected by SYBR® Green I.
    • What is the difference in sensitivity between TaqMan® chemistry vs. SYBR® Green reagent chemistry?
      Sensitivity is equivalent when using TaqMan® chemistry and SYBR® Green reagent chemistry. Since a fluorescent signal is generated by a sequence specific TaqMan® probe, users might think a TaqMan® assay is more sensitive than a SYBR® Green reagent assay. This is not always true. A poorly designed TaqMan® assay could theoretically be less specific than a well-designed SYBR® Green reagent assay. The potential for detection of primer dimers and non-specific products using SYBR® Green reagent chemistry may result in loss of sensitivity when attempting to quantitate lower copy numbers.
    • What is the concentration of my primers and TaqMan probe to be used with Taqman assays?
      Optimal results may require titration of primer concentration between 100 and 900 nM. A final concentration of 300 nM each primer and 100 to 250 nM probe is effective for most applications. However, increasing the concentration of the primer that initiates synthesis of the target strand that is complementary to the probe can improve fluorescent signal for some primer/probe systems.
    • How should I store my cDNA?
      The cDNA can be diluted in low EDTA TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA) or water and stored at 4 C or at -20 C.
    • How much cDNA should be used in each qPCR reaction?
      You can use from 10 ng down to 0.1 pg of cDNA in each qPCR reaction. The kit provides maximum flexibility with regards to the amount of starting RNA and the amount of cDNA used in the qPCR. The microRNA cDNA can be diluted appropriately to accommodate the amount of total RNA used in the cDNA synthesis reaction and the relative abundance of the microRNAs of interest. We recommend starting with 1 ng of cDNA in each qPCR. If an individual microRNA is relatively abundant then 0.1 ng may work fine. If the microRNA is rare or absent you can add up to 10 ng of cDNA to the qPCR reaction. In this case we recommend comparing your results to a no-poly(A) polymerase reaction. A significant difference between reactions with and without poly(A) polymerase will help determine if the sample is positive or negative for the microRNA of interest. Three examples are provided below: For abundant microRNAs where total RNA sample is limiting: • 20 ng of total RNA in a 20 uL cDNA synthesis reaction = 1 ng /uL • Dilute with TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to 0.02 ng/uL (1/50 dilution) • Add 5 uL (0.1 ng) to a 20 uL qPCR • 200 total qPCRs For rare microRNAs: • 200 ng of total RNA in a 20 uL cDNA synthesis reaction = 10 ng/uL • Dilute with TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to 2 ng/uL (1/2.5 dilution) • Add 5 uL (10 ng) to a 20 uL qPCR • 20 total qPCRs For profiling experiments: • 1000 ng total RNA in 20 uL cDNA synthesis reaction = 50 ng/uL • Dilute with TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) to 0.2 ng/uL (1/250 dilution) • Add 5 uL (1 ng) to a 20 uL qPCR • 1000 total qPCRs
    • What is the function of the antibody in AccuStart Taq Polymerase? How does this contribute to hot start PCR?
      AccuStart Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with proprietary antibodies that inhibit polymerase activity. Due to specific binding of the antibodies, the activity of Taq DNA polymerase is blocked at ambient temperatures. Therefore, AccuStart Taq Polymerase remains inactive during reaction assembly and initial temperature ramp up. Antibodies are inactivated at the initial PCR denaturation step and release fully active Taq DNA Polymerase. This process provides an automatic hot start and improves PCR specificity, sensitivity and yield significantly. It also allows room temperature reaction assembly for high though put applications. By increasing the effectiveness of Taq DNA polymerase through the use of this product, it is possible to reduce the optimization and handling of reaction components and improve PCR results.
    • Stability of Taq polymerase
      The AccuStart Taq Polymerase and antibody should be stable a minimum of 18 months if stored properly at -20C in a non frost free freezer.
    • Components of Taq buffer
      The Accustart Taq PCR Buffer is supplied as a 10X concentrate and should be diluted 1:10 (1 part buffer + 9 parts other components = 10 parts final reaction volume). Buffer Composition (10X): 200 mM Tris-HCl (pH 8.4), 500 mM KCl. The Taq PCR buffer does not contain Magnesium Chloride. This is provided in a separate tube as 50 mM Magnesium Chloride
    • Units of AccuStart Taq DNA polymerase to be used in PCR
      1-1.5 units per 50 ul reaction are sufficient for most applications, but in some instances it may be necessary to use more enzyme.
    • DMSO inhibition of Taq DNA polymerase
      Taq DNA polymerase is inhibited 47% at a DMSO concentration of 10%. 10% DMSO is used in PCR or cycle sequencing reactions of GC rich DNA in the presence of 2X the amount of Taq DNA polymerase (see Sun, (1993) BioTechniques)
    • Does Taq DNA polymerase have RT activity
      Taq DNA Polymerase has some reverse transcriptase (RT) activity at 68-78°C. However, this activity is negligible under ordinary PCR conditions. We don’t recommend using Taq Polymerase as a reverse transcriptase.
    • Benefits of AccuStart Taq DNA Polymerase compared to regular Taq
      AccuStart Taq DNA Polymerase is a recombinant Taq DNA polymerase preparation which contains monoclonal antibodies that bind to the polymerase and keep it inactive before PCR thermal cycling. It allows for automatic hot start PCR, without extra work by the scientist:  - Room temperature reaction assembly.  - Broader optimal Magnesium concentration.  - Less primer optimization. Upon heat activation (1 minute at 94ºC), the antibodies denature irreversibly, releasing fully active Taq DNA polymerase. Non-specific extension of primers at low temperatures is a common cause of artifacts and poor sensitivity in PCR. The AccuStart automatic hot-start enables specific and efficient primer extension in the PCR process with the added convenience of room temperature reaction assembly. Activated AccuStart Taq DNA polymerase possesses 5’→3’ DNA polymerase activity and a double-strand specific 5’→3’ exonuclease. The polymerase does not have 3’-exonuclease activity and is free of any contaminating endo or exonuclease activities. One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 74°C. AccuStart Taq DNA polymerase is stable for 2 years when stored in a constant temperature freezer at 20ºC.
    • Does AccuStart Taq DNA Polymerase remain in an active state once it is activated?
      Yes, once activated, AccuStart Taq DNA Polymerase remains active. Lowering the temperature will not inactivate AccuStart Taq DNA Polymerase.
    • Unit definitions for AccuStart Taq DNA Polymerase
      One unit (U) of enzyme is defined as the amount that will incorporate 10nmoles of dNTPs into acid insoluble material per 30 minutes in a 10-minute incubation at 74 oC under the analysis conditions provided in the product insert. AccuStart Taq DNA Polymerase is premixed with anti Taq Antibodies. It can be activated by heating at 95C for 1-3 minutes.
    • Does AccuStart Taq DNA Polymerase have proofreading activity?
      No, AccuStart Taq DNA Polymerase does not have proofreading activity. Fidelity of this Polymerase in PCR amplifications may be improved, by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.
    • What is the activation time for AccuStart™ Taq DNA Polymerase and AccuFast™ Taq DNA Polymerase ?
      "The recommended activation time depends on which Supermix is being used: Activation Conditions for AccuStart™ Taq DNA Polymerase in: PerfeCTa qPCR Supermixes 1-2 min, 95C PerfeCTa SYBR Green Supermixes 1-2 min, 95C Activation Conditions for AccuFast™ Taq DNA Polymerase in: PerfeCTa qPCR FastMixes 20 sec, 95C PerfeCTa SYBR Green FastMixes 20 sec, 95C

Publications

  • PCR
    • Real-Time Quantitative PCR
      • SYBR Green Detection
        • DNA
          Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems
          Daniel Rodrigues - 2022
          Abstract
          Background T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. Methods GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. Results Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. Conclusions Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.
          Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit
          Larissa Bessler - 2022
          Abstract
          Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
      • Probe-based Detection
        Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring
        Rose Schrott - 2022
        Abstract
        Background Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable. Results Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers. Conclusions This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a “washout” period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly. These results support that paternal preconception exposure to cannabis can influence offspring outcomes.
        Experimental challenge of flatfishes (Pleuronectidae) with salmonid alphavirus (SAV): Observations on tissue tropism and pathology in common dab Limanda limanda L.
        Linda Andersen - 2022
        Abstract
        Salmonid alphavirus (SAV) is the aetiological agent of pancreas disease (PD), a serious viral disease in salmonids. For several decades, SAV was known to infect salmonid species only, until SAV was detected using real-time PCR in several species of wild-caught flatfishes in Scotland in 2010. The presence of SAV in wild flatfishes has been confirmed by further surveys from Ireland and Scotland. The role of flatfishes in SAV-spread and epizootiology has not been elucidated, and no experimental challenges have been conducted to examine virus tissue tropism, virulence and pathology in flatfishes. Wild-caught flatfishes (common dab; Limanda limanda, European plaice; Pleuronectes platessa, European flounder; Platichthys flesus and lemon sole; Microstomus kitt) were either intramuscularly (i.m.) or intraperitoneally (i.p.) challenged with SAV3 or exposed to SAV3 through cohabitation with i.p. injected salmon. SAV-infections were seen in i.m. and i.p. injected dab and i.p. injected salmon but did not result in a transmissible infection in dab although several routes of entry were assessed (oral route not tested). SAV was detected in several tissues of eight common dab (not from cohabitants), with high SAV-levels in pancreas. No viraemia was detected in the SAV-positive common dab and no virus shedding were detected in the tanks. However, pathology in exocrine pancreas and hearts consistent with SAV-replication were seen. This is the first study reporting SAV-induced pathology in a non-salmonid species. The results from the present challenge study supports evidence for common dab being susceptible hosts for SAV. The study also demonstrates that flatfishes are less susceptible to SAV3-infection than salmon.
        Validation of Microchip Based RT-PCR ABC Test (InfA/B & COVID-19) in Clinical Samples
        Gabriel Martinez - 2022
        Abstract
        To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.
        Efficiency-corrected PCR quantification for identification of prevalence and load of respiratory disease-causing agents in feedlot cattle
        RJ Barnewall - 2022
        Abstract
        Bovine respiratory disease (BRD) is the most prevalent disease in feedlot cattle worldwide with Bovine alphaherpesvirus 1 (BoAHV1), Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida and Trueperella pyogenes accepted to be common etiological agents associated with BRD. Although these agents are common in the upper and lower airways in clinical BRD cases, some also exist as normal flora suggesting their presence in the upper airways alone is not necessarily informative with respect to disease status or risk. To determine the relationship between presence, load and disease status, we investigated the relationship between load in the upper airways at induction and active BRD cases in feedlot cattle using efficiency-corrected PCR quantification. By this approach, we were able to accurately determine the prevalence and load of the key BRD agents in the upper respiratory tract showing that cattle in the hospital pen had a higher prevalence, and load, of these agents both singly and in combination compared to cattle sampled at feedlot induction. A combination of agents was the most accurate indicator of BRD risk with cattle with four or more agents detected in the upper airway more likely to be undergoing treatment for BRD than non-BRD ailments. In addition, M. bovis was rarely detected at feedlot induction but was identified at high prevalence in cattle in the hospital pen. These findings present a potential new technological approach for the investigation, analysis and identification of BRD-associated viral and bacterial agents for Australian feedlot systems as well as for BRD disease management and treatment
        Systematic stepwise screening of new microbial antagonists for biological control of European canker
        G. Elena - 2022
        Abstract
        Neonectria ditissima is the causal agent of European canker. This pathogen causes cankers on apple branches and the main stem, which may lead to the loss of the whole tree. Chemical control is the essential component in disease management and no suitable biocontrol products are commercially available. This study aimed at selecting potential microbial antagonists against N. ditissima through a systematic stepwise screening program for the development of a new biocontrol product. A total of 520 potential candidates were isolated from apple branches showing canker symptoms. Important characteristics for application of Microbial Biological Control Agents were tested per each candidate: spore production, spore survival during storage, cold tolerance, drought tolerance and UV-B resistance. Isolates able to germinate and grow at human body temperature were excluded. A total of 252 candidates fulfilled the stablished criteria. All 520 candidates belonged to 44 different taxonomic groups, being the most abundant Alternaria spp. (22.2%), Aureobasidium pullulans (16.1%), Cladosporium spp. (9.5%) and Fusarium spp. (9.0%). Information on possible pathogenicity and toxicity for humans, animals, plants and the environment and on patents in biocontrol use was collected per each identified species. A total of 158 candidates belonging to species that did not show potential risks or patent conflicts were assessed by their antagonistic behaviour against N. ditissima in bioassays in planta. Each candidate was inoculated in ‘Elstar’ apple branches inoculated with N. ditissima 24 h before. Inoculated branches were incubated at 17 °C, 16 h light per day and RH>90%. After four weeks, canker symptom expression was visually assessed. The capacity of the candidates to reduce colonisation of N. ditissima in the branches was evaluated by quantifying N. ditissima DNA concentration using qPCR. Four candidates of Clonostachys rosea showed antagonistic properties; after four weeks of treatment, no canker symptoms or small bark cracks were observed in the inoculated branches and N. ditissima DNA was reduced by 90-99%. Following them, the branches inoculated with one candidate of Akanthomyces muscarius, A. pullulans and Cladosporium europaeum showed small bark cracks and small swollen bark and N. ditissima DNA was reduced by more than 90%. The systematic stepwise screening approach was a powerful strategy to find new MBCAs against N. ditissima with antagonistic properties that also fulfilled major criteria with regards to commercial production and safety, as well as ecological needs.
        Engineering heterologous enzyme secretion in Yarrowia lipolytica
        Weigao Wang - 2022
        Abstract
        Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and α-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.
        TIRAP/Mal Positively Regulates TLR8‐Mediated Signaling via IRF5 in Human Cells
        Kaja Elisabeth Nilsen - 2022
        Abstract
        Toll‐like receptor 8 (TLR8) recognizes single‐stranded RNA of viral and bacterial origin as well as mediates the secretion of pro‐inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll‐inter‐ leukin 1 receptor domain‐containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte‐derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine‐gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, re‐ spectively, while nuclear translocation of transcription factors was addressed by immunofluores‐ cence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt‐kinase acti‐ vation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5‐dependent cyto‐ kines IFNβ and IL‐12p70 as well as, to a lesser degree, TNF. These findings reveal a new and un‐ conventional role of TIRAP in innate immune defense