Rotor-Gene 6000 Series Software

CRISPRi is not strand specific at all loci and redefines the transcriptional landscape. Reviewer 1 In recent years, transcriptional interference by antisense RNA transcription has been recognized as a relatively frequent means to regulate m. RNA expression. Tools enabling antisense transcription arrest without directly affecting sense transcription are required to study the mechanism mediating this effect. The recently reported CRISPRi system, using a modified, partially defective, Cas. Cas. 9 appeared as ideally suited for this since it has been shown to be able to trigger transcription termination in a site specific and, most importantly, strand specific manner, without altering the genomic sequence. CRISPRi, an adapted CRISPRCas9 system, is proposed to act as a strandspecific roadblock to repress transcription in eukaryotic cells using guide RNAs sgRNAs to. This short report shows that this strand specificity is actually not true at all loci. In addition, it describes an example in which the binding of the guide RNA actually induces spurious transcription initiation, suggesting that it markedly perturbs the local chromatin environment. I think that the data are suitably convincing. Note, by the way, that this referee has made the similar unpublished observations that the termination by this system was not strand specific at yet another locus. Although one could argue that the main results presented in this manuscript are negative results, I personally think that they are well worth being published to warn further investigators who would wish to use this system and a short report in e. Life might seem appropriate for that. We thank the reviewer for recognising the importance of this work as a warning to other researchers. The position of the 5 end of SUT6. Indeed, if one refers to the TIF seq data from the original Pelechano paper, the main transcription start site for SUT6. Crick strand of chromosome X this is also consistent with the data from Malabat et al., e. Life, 2. 01. 5 would be located about 6. DOWNSTREAM of the AS 4. NT sg. RNA binding site position 7. Thus, as the other SUT6. RNA targeting sequences, AS 4. NT would actually be located within the SUT6. TSS and not upstream. It could thus not be considered as a control sg. RNA. Could the authors verify this point and, if this is correct, modify the text, Figure 2. E and Figure 2figure supplement 1 accordingly This would in no way modify the conclusions of the manuscript. Table of Contents Foreword 0 Part IDisclaimer 5 Part IIIntroduction 5 1Welcome. E2%80%99s+only+real-time+rotary+thermo-optical+analyser.jpg' alt='Rotor-Gene 6000 Series Software' title='Rotor-Gene 6000 Series Software' />We thank the reviewer for pointing this out and apologise for our oversight. We note that there is a large heterogeneity in the position of the transcription start site for SUT6. Nguyen et al., 2. We have now discussed the transcript heterogeneity in the main body of the text and included a supplementary figure with the Pelechano TIF seq data to illustrate this Figure 2figure supplement 1. We have also renamed the HMS2 strains in the text and figures based on the new AS targeting sg. RNA positions relative to the major SUT6. TSS at position 7. Reviewer 2 In this paper by Howe et al., the authors assess the potential of CRISPRi for strand specific transcriptional perturbation in budding yeast. They study two genes in depth GAL1 and HMS2 and compare the effectiveness of CRISPRi to previous approaches for example deletion of cis acting motifs such as the TATA box in wt and Xrn. Their primary conclusion is that CRISPRi works well in some cases and not in others and requires careful controls. For example, the authors show that the system does repress antisense transcription, but at one of the two studied loci HMS2, an additional antisense transcript appears from a new initiation site, and the sense transcript is prematurely terminated. In addition, they present a clever tool based on click chemistry for quickly making g. RNA. Although I dont find the main conclusion revelatory, I commend the authors for their thoroughness. Certainly, this message is one that bears reporting, although I am not quite convinced this result clears the bar for an e. Life paper. We thank the reviewer for their support of the message we are trying to convey to the community. We feel that e. Life, with its reputation for thoroughness and research integrity, would be an ideal journal for publishing this work. The authors show that CRISPRi represses the antisense transcript at a GAL1 model gene. They then check whether this repression has transcriptional effects on GAL1 sense transcription and they conclude there is no difference in galactose. However, previous work has shown that antisense at the GAL locus works mostly under repressive conditions. Have the authors repeated this experiment in glucose or raffinose As presented in Figure 3. Rpg Maker Vx Ace Monster Graphics. A, we observed no leaky expression of GAL1 sense transcript in glucose t0 in the absence of the antisense and the sense induction kinetics are similar to the control. Rotor-Gene 6000 Series Software' title='Rotor-Gene 6000 Series Software' />Quantitative PCR protocol using SYBR Green reagents. A variety of reagents provided to meet users needs for multiple instruments and applications. A continuacin le ofrecemos un resmen ampliado, de algunos productos de equipamiento auxiliar analtico. No pretende ser de ningn modo, un resmen completo. REST 2008 is a standalone software package for analyzing gene expression using realtime amplification data. The software addresses issues surrounding the measurement. BIOLOGA MOLECULAR. Deteccin de la mutacin V617F del gen JAK2 mediante anlisis de disociacin de alta resolucin. Detection of V617F mutation of JAK2 gene. We have included a sentence in the manuscript text to explain this. The authors show that CRISPRi repression at the HMS2 locus results in a changed transcriptional landscape. Replacement of the HMS locus by URA does not have the same effect, and they suggest that CRISPRi binding causes these additional transcripts. However, it is unclear whether the new transcripts at HMS2 appear because of removal of the antisense, which in itself could change the chromatin landscape and thus the transcriptional landscape, or whether it arises because of binding of d. Cas. 9. The same authors have previously published that removal of the GAL1. RNA by genetic methods point mutations of the transcription factor binding sites also results in additional transcripts not visible in the wildtype Murray et al., 2. S5. C. Given these findings, it remains unclear whether the new antisense transcript indeed results from the CRISPRi binding. Moreover, it is noteworthy that additional transcripts are also found in some genetic mutants. Control experiments are therefore required for both methods. The point made here by this reviewer is a valid one we are unable to infer whether it is the binding of sg. RNAd. Cas. 9 or the loss of the antisense that is causing the initiation of the new transcripts. It would be experimentally very difficult to show this and we have now clarified the two possible reasons for new transcripts in the text. However, the main point is that, independent of what is causing the initiation of the new antisense transcript, in the CRISPRi strain, transcription is occurring in the antisense direction into the HMS2 sense promoter and so no conclusions could be made about the function of SUT6. The fear is that some investigators may use the CRISPRi system without the proper controls and the current set of experiments is intended to highlight how important these controls are. The Xrn. 1 sensitivity is a confounding factor, and I am not sure that strain should be the gold standard for CRISPRi not working. The whole point is that one can get blocking or perturbation in the absence of genetic manipulation. Rotor-Gene 6000 Series Software' title='Rotor-Gene 6000 Series Software' />So, for example one concluding sentence CRISPRi is not as effective as a genetic mutation in reducing levels of either the GAL1 or HMS2 AS transcripts should end with in the Xrn. We apologise that we were not clear about the reason for performing the experiments in the XRN1 delete and have now clarified this in the text. We chose to use the deletion of XRN1 to stabilise any transcripts that may be being produced in the CRISPRi strains but then rapidly degraded so that, if the experiment is done in an XRN1 background, the transcript could not be detected. Universal SYBR Green Quantitative PCR Protocol. Technology Overview SYBR based Quantitative PCRWith the development of thermal cyclers incorporating fluorescent detection, PCR has a new, innovative application. In routine PCR, the critical result is the final quantity of amplicon generated after the process. Real time or Quantitative PCR and RT PCR use the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation. In quantitative PCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the Quantification Cycle Cq or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against Cq. The amount of DNA or c. DNA in an unknown sample can then be calculated from its Cq value. A The different phases of the reaction Baseline The initial concentration of template is low therefore, the fluorescence intensity is too low to be detected and only the background signal is evident. Exponential After the target yield has reached the detection threshold, shown as the red threshold line, the course of the reaction can be followed through the exponential phase. Linear As the concentration of template increases, the available DNA polymerase concentration reduces and the reaction rate decreases. Plateau There is insufficient free enzyme to continue amplification and so after this point, the reaction is at the maximum yield, or the plateau phase. B Individual reactions are characterized by the cycle at which fluorescence first rises above the threshold, which is referred to as the Quantification Cycle Cq. If the starting material is abundant, amplification is observed in earlier cycles, and the Cq is lower. If the starting material is scarce, amplification is observed in later cycles, and the Cq is higher. This correlation between fluorescence, Cq, and amount of amplified product enables quantification of the template over a wide dynamic range. Real time PCR also lends itself to relative studies. A reaction may be performed using primers unique to each region to be amplified and tagged with different fluorescent dyes. Several commercially available quantitative thermal cyclers include multiple detection channels. In this multiplex system, the amount of target DNAc. DNA can be compared to the amount of a housekeeping sequence e. GAPDH or actin. SYBR Green q. PCR Applications Mass Screening. XXMicroarray Validation. XXMultiple target genesfew samples. XSNP detection. NRAllelic discrimination. NRPathogen detection. XMultiplexing. NRViral load quantification. NRGene expression analysis. XGene copy determination. NREnd point genotyping. NRin vitro quantification. NRNR not recommended. X recommended. XX preferred method. For additional information, please see the q. PCR Technical Guide or SYBR Green I based q. PCR technical animation. Assay Considerations. DNA Preparation. The single most important step in assuring success with PCR is high quality DNA preparation. Integrity and purity of DNA template is essential. Quantitative PCR involves multiple rounds of enzymatic reactions and is therefore more sensitive to impurities such as proteins, phenolchloroform, salts, EDTA, and other chemical solvents. Contaminants can also interfere with fluorescence detection. The ratio of absorbance values at 2. DNA purity. Pure DNA has an A2. A2. 80 ratio of 1. Lower ratios indicate the presence of contaminants such as proteins. Template. Very few copies of target nucleic acid equivalent to about 1. DNA or c. DNA are needed to initiate q. PCR. To minimize contamination with reaction inhibitors, the starting template amount should be kept to the minimum required to achieve accurate quantification. When the starting material is RNA, primer design and DNase I treatment will reduce signals that may be generated from g. DNA contamination. Primer Design. Whether using a ds. DNA binding dye or a probe based detection chemistry, designing high quality primers is one of the most crucial pre experimental steps in q. PCR. Specific primers for PCR should be designed with the aid of primer design software to eliminate the complications introduced with primer dimers and secondary structures. Lower primer concentrations decrease the accumulation of primer dimer formation and nonspecific product formation, which is critical in using SYBR Green I dye in quantitative PCR. NTPs. Standard PCRq. PCR mastermixes contain d. ATP, d. CTP, d. GTP, and d. TTP. However, some mixes are available that replace d. TTP with d. UTP. Products from previous reactions run with d. UTP will contain uracil instead of thymine. These are then susceptible to cleavage by Uracil DNA Glycosylase UNG. Therefore, prior incubation of subsequent reactions with UNG prevents carry over contamination between reactions. To be effective, all reactions in the laboratory must use d. UTP. Magnesium Concentration. Magnesium chloride Mg. Cl. 2 is necessary for reverse transcriptase, Taq DNA polymerase, and Taq DNA 5 to 3 exonuclease activity. Optimum Mg. 2 concentrations for reactions containing DLP are usually between 3 6 m. M. Lower magnesium chloride concentrations usually result in the formation of fewer nonspecific products. Some Ready. Mix solutions are provided at a 2. X concentration of 7 m. M magnesium chloride final concentration 3. M. In some cases, a vial of a 2. M magnesium chloride solution is provided for further optimization of the final magnesium chloride concentration if necessary. A reaction mix that does not contain Mg. Cl. 2 may sometimes be required so that a low concentration can be used, e. Scorpion Probe detection. Reverse Transcriptase. A reverse transcriptase enzyme that provides high yields of c. DNA, while retaining activity at high temperature, is critical to the success of RT q. PCR. Performance at high temperatures helps to ensure that regions of RNA with significant secondary structure are destabilized and accessible for hybridization and subsequent amplification. When performing one step RT q. PCR, high temperature performance allows the use of gene specific primers with high melting temperatures Tm, which increases reaction specificity. When performing two step protocols, it is important to ensure that the enzyme results in a linear and proportional yield of c. DNA from RNA. Minimizing pipetting can decrease variability. Some Ready. Mixes contain primers and other reagents needed to perform RT, for example, Ready. Script c. DNA Synthesis Mix RDRT. Taq DNA Polymerase. As with selecting the most appropriate reverse transcriptase for the RT, selection of the appropriate enzyme is vital. A fundamental problem with natural Taq DNA polymerase is that the enzyme has residual activity at low temperature. Non specific primer binding leads to non specific product formation as a result of this residual polymerase activity. Antibody blocked or chemically blocked Taq DNA polymerases hot start help to rectify this situation by preventing enzyme activity until the high temperature, denaturation step begins. Refer to the PCR Mix Selection Guide to define the best hot start polymerase for your application. Internal Reference Dye by Instrument Type.