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Sample PreparationWhat is the shelf life of the Extracta DBS product?The shelf life is 2 years stored at 2-8°C.What is the Extracta DBS kit and what are some of the advantages of using this kit?The Extracta DBS kit is a research use only reagent for rapid isolation of DNA from dried blood spots. It is supplied as a convenient “ready to use” solution that reduces sample preparation time and is compatible with automation. The numerous wash and/or proteolytic digestion steps of other protocols are not necessary with Extracta DBS and the eluted DNA solution does not interfere with downstream DNA amplification applications. Qiagen Beverly’s ISO13485 manufacturing process ensures consistent composition and performance across lots.Should all of the supernatant be removed after the first wash and centrifugation step?After the first wash and centrifugation step, remove and discard as much liquid and debris as possible. The DNA remains on the filter paper at this point.What Quantabio products have confirmed compatibility with DNA extracted by the Extracta DBS kit?The following Quantabio products have been demonstrated to perform well with the extracted DNA: Accustart Genotyping ToughMix (95115, 95116, 95117) PerfeCTa Multiplex qPCR ToughMix (95147, 95148, 95149) PerfeCTa qPCR ToughMix (95112, 95113, 95114, 95138, 95139, 95140)For what downstream applications can the extracted DNA be used?The extracted DNA is compatible with a variety of end-point PCR, real-time PCR, next generation sequencing, or Sanger sequencing procedures.Can the extracted DNA be stored?We recommend using the DNA in downstream applications immediately after extraction. If this is not possible, the extracted DNA can be refrigerated at 2-8°C for short term storage (1-2 days) or stored frozen at -15 to -25°C for prolonged storage. We have no data on the stability of extracted DNA after prolonged storage.What is the stability of the extracted DNA?We have no data on the stability of the extracted DNA. We recommend that the DNA be used for downstream applications as soon as possible following extraction.How should the extracted DNA be measured?Measurement of DNA by methods using DNA binding dyes or absorbance at 260 nm are not applicable to a crude lysis method such as the Extracta DBS method. The recommended way to accurately quantify DNA in an Extracta DBS lysate is to measure a specific DNA sequence (generally a single-copy gene in gDNA) by qPCR.
Reverse TranscriptionFirst-Strand cDNA SynthesisIs MMLV RT as stable in the qScript cDNA Supermix as in the qScript cDNA synthesis kit? Do you recommend storing these products at -80C?In the qScript cDNA synthesis kit, the RT/RNase inhibitor mix is provided as a concentrated enzyme separately from the 5X reaction mix. This enzyme is actually stable at -20C for a year. The cDNA SuperMix has a shelf life of 12 months at -20C. Neither product shows any diminished performance after storage in a REVCO at -70C to -80C; two years for the qScript cDNA Supermix and five years for the qScript cDNA synthesis kit. If you are looking to prolong shelf life, storage at -80C is best for the qScript cDNA Supermix. Storage at -20C is sufficient for the qScript cDNA Synthesis Kit.I am trying to amplify a very rare transcript. Can I scale up a cDNA synthesis reaction and use 2ug of mRNA?qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. For the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL. Simply scale each component proportionally.Does the qScript™ cDNA Synthesis Kit include an RNase Inhibitor like the qScript™ cDNA SuperMix?Both kits contain an RNase inhibitor protein. qScript™ cDNA SuperMix is provided as a single tube reaction of 5X concentrated master mix. It provides all necessary components for first-strand synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers (except RNA template) . In the case of the qScript™ cDNA Synthesis Kit, Rnase inhibitor is premixed with the reverse transcriptase and is provided as a concentrated enzyme.Do the cDNA Supermix and the cDNA synthesis Kits have the same dynamic range in RNA quantity?Both qScript cDNA Supermix and qScript cDNA Synthesis Kits have a broad linear dynamic range. We recommend using them for first-strand cDNA synthesis using HeLa cell total RNA dilutions ranging from 1 µg to 1 pg. One tenth of each first-strand reaction should be used for qPCR amplification. Within this range of HeLa total RNA, qPCR results should be equivalent with both kits. qScript cDNA synthesis kit may have advantages over the qScript cDNA Supermix in certain situations such as working with small quantities of RNA. qScript cDNA Supermix outperforms the qScript cDNA Synthesis kit when starting with quantities of total RNA in excess of 100 ng. qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. With the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL and simply scaling each component proportionally.What is the qScript™ Flex cDNA Kit? How is it different from the qScript™ cDNA Synthesis Kit?The qScript Flex cDNA Synthesis Kit is an easy-to-use and highly efficient kit for the synthesis of first-strand cDNA that enables your choice of cDNA priming method. Various RT-PCR applications may require different priming strategies for optimal performance and this kit provides optimized reagents for priming with oligo(dT)20, random primer, gene-specific primer (GSP), or any combination thereof. Therefore, qScript™ Flex cDNA Kit is ideal for developing the best protocol for your application. The qScript™ Flex cDNA Kit also allows the use of Oligo dT or gene-specific primers (GSP) for long RT-PCR of RNA targets up to 12 kb. The qScript cDNA Synthesis Kit consists of an optimized blend of random and oligo(dT) primers. It provides robust, consistent and unbiased first-strand synthesis over a broad range of RNA template concentrations. qScript™ cDNA Synthesis Kit is well suited for short RT-PCR apllications (i.e. qRT-PCR) and is not recommended for amplification of RNA's longer than 1kb. When using a mixture of the Oligo dT and Random Primer solutions, the qScript™ Flex cDNA Kit achieves first-strand synthesis over a broad range of RNA template concentrations and its performance is comparable to that of the qScript™ cDNA Synthesis Kit. With both kits, the resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR.What is the difference between qScript cDNA SuperMix and the qScript cDNA Synthesis kit?qScript cDNA SuperMix is the first ""true"" SuperMix format commercially available for cDNA synthesis. A single tube reaction, of 5X concentrated master mix, provides all necessary components for first-strand synthesis (except RNA template) including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers. In the qScript cDNA Synthesis kit the qScript RT (mixture of RNase inhibitor protein and qScript reverse transcriptase) is provided as a concentrated enzyme. A separate tube contains a solution of 5X concentrated reaction buffer, dNTPs, MgCl2, and primers. Aside from the inclusion of the enzyme and the stabilizers in the qScript cDNA SuperMix, its formulation is also slightly different from the qScript cDNA Synthesis formulation.What is the Flex Kit? How is it different from the cDNA synthesis Kit? Can we use either an oligo dT or a random priming method?The Flex Kit supports the use of multiple priming strategies. When using a combination of the oligo dT primer solution and random primer solution, the linear dynamic range and qRT-PCR performance is comparable to that of the qScript cDNA Synthesis Kit. In addition to retaining high performance with limiting amounts of RNA, the Flex kit was also optimized to provide long cDNA synthesis with oligo dT primer. So its composition is different from that of the cDNA synthesis Kit. When using oligo dT primer alone, it is unquestionably one of the easiest to use and most sensitive cDNA kits on the marketFor first strand cDNA synthesis, is it better to use oligo(dT), random hexamers, gene specific primer (GSP) or combination of these primers?The choice of primer depends on your experimental goals. Oligo(dT) is recommended when using total RNA for cDNA synthesis. It is the key to full-length cDNA synthesis. Random hexamers give a series of short first-strand products spanning the entire mRNA. Use of random hexamers may be helpful if the PCR fragment is at the 5´ end of a large mRNA. To ensure full-length cDNA synthesis of large transcripts, oligo(dT) can be added along with random hexamers during first-strand synthesis. Gene specific primers (GSP) for cDNA synthesis may also be used and are required in a few applications such as 5´ RACE and qRT-PCR. For GC-rich templates, or templates rich in secondary structure, a GSP may not work as well as priming with oligo dT for first strand synthesis. If an RT-PCR is problematic, trying different options of oligo dT, random primers and/or GSP for priming first strand synthesis may find a solution. Oligo(dT)20 primer (Cat. No. 18418-020) is recommended for use with SuperScript III Reverse TranscriptaseWhat is the smallest quantity of RNA detectable by the qScript™ cDNA Synthesis Kit?Lower limits of detection by the qScript™ cDNA Synthesis Kit are dependent on many factors, such as primer design, target size, and the abundance of message. However, this system was able to detect GAPDH mRNA from as little as 1.0 pg of total HeLa RNA when used in conjunction with AccuStart Taq DNA Polymerase.What is the RNA input for the qScript cDNA Synthesis Kit?The qScript cDNA Synthesis Kit provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.What is the RNA input for qScript cDNA Supermix?The qScript cDNA Supermix provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.In conventional cDNA-synthesis, an RNA-denaturation step is often included (typically 65 degrees C for 15 min.). Such a step is not mentioned in the protocol for the qSript cDNA SuperMix, is it not needed?We have not seen any need or benefit to including an RNA denaturation step with the qScript cDNA SuperMix. We have examined hundreds of different amplicons using real-time RT-PCR and have not seen any difference. However, there are exceptions to every rule – especially when it comes to RT-PCR. Disruption of RNA secondary structures, immediately prior to carrying out cDNA synthesis, is critical when using oligo-dT or gene-specific priming. Inclusion of the denaturation step is also important when using oligo-dT primer for generating long first strand products (i.e. amplification of full length genes). Our qScript Flex cDNA Synthesis kit allows the flexibility to choose the method of your choice (oligo-dT, random primer, gene-specific primer, or any combination thereof) for priming first-strand synthesis. We have been able to generate cDNAs that were over 15-kb long with this kit. How the denaturation step is carried out is very important. Often, incubation of RNA and primer in water alone at elevated temperatures can result in non-specific hydrolysis and degradation of the RNA. The qScript Flex kit includes a special additive in the primer solutions, as well as a separate tube of this enhancer solution for use with gene-specific primers, that protects the RNA from hydrolysis and facilitates efficient annealing of the primer(s). In the case of the cDNA SuperMix, it is possible that annealing of random primers to the RNA may facilitate disruption of secondary structure, and thus obviate the need for the denaturation step. Links to product information: qScript™ cDNA Synthesis, qScript™ cDNA, qScript™ Flex cDNA Links to product information: qScript™ cDNA Synthesis Kit:http://www.quantabio.com/pdf/manuals/95047%20(qScriptT%20cDNA%20Synthesis%20Kit%20PPS).pdf qScript™ cDNA SuperMix:http://www.quantabio.com/pdf/manuals/95048%20(qScript%20cDNA%20SuperMix%20PPS).pdf qScript™ Flex cDNA Kit:http://www.quantabio.com/pdf/manuals/95049%20(qScriptT%20Flex%20cDNA%20Synthesis%20Kit%20PPS).pdfReverse Transcription PCR (RT-PCR)Quantitative RT-qPCRWhat is the amount of RNA used per TaqMan® or SYBR qPCR Assay?Suggested input quantities of template are: 1 pg to 1 μg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. For more information, please consult the qScrip One-Step qRT-PCR Kits protocols.
PCR & qPCRReal-Time Quantitative PCRSYBR Green DetectionCan I use PerfeCTa® SYBR® Green FastMix® instead of SYBR Green SuperMix?You can use FastMix instead of SuperMix. We have observed similar results with both PCR mixes, however, slightly lower background was observed using SuperMix when using 10 ng of cDNA or more in the qPCR. The qScript microRNA Quantification system has been validated using SYBR Green SuperMix.What is the amount of cDNA used per TaqMan or SYBR qPCR Assay?Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 100 pg to 100 ng genomic DNA. For more information, please consult the PerfeCta qPCR Supermixes or PerfeCta SYBR Green Supermixes protocols.Is there a SYBR® Green Fast Master Mix currently availableYes. PerfeCTa SYBR Green Supermix is available. For further information, please consult the Kit protocol or product information.Probe-based DetectionWhat are the advantages of PerfeCTa qPCR FastMix vs PerfeCTa qPCR Supermix?Amplification can be run in about X minutes, at least 3X faster than standard runs with the PerfeCTa qPCR Supermix, with comparable results. For more information on using these reagents, please consult the PerfeCTa qPCR FastMix and PerfeCTa qPCR Supermix Protocol.Storage of PerfeCTa qPCR FastMixWe recommend a storage temperature of 2 – 8 ºC for the TaqMan® Fast Universal PCR Master Mix (P/N 4352042). The shelf life is 9 months from the manufacturing date.SNP assays with PerfeCTa qPCR FastMix?The TaqMan® SNP Genotyping assays have not been fully tested on the Applied Biosystems 7500 Fast System.What component in the PerfeCTa qPCR FastMix allows rapid amplification results?The hot-start DNA polymerase enzyme system in the PerfeCTa qPCR FastMix only requires a 30 seconds activation step, and supports significantly shorter hold times for the thermal cycling stages of PCR, thereby minimizing PCR run time. For more information on using this reagent, please consult the PerfeCTa qPCR FastMix (2X) protocol.How do assays performed with PerfeCTa qPCR FastMix compare with assays performed with PerfeCTa qPCR FastMix?In general, assays have been found to perform comparably. Please consult the PerfeCTa qPCR Supermix (2X) Protocol for more information on using this reagent.What are the applications for the PerfeCTa qPCR FastMix?Absolute Quantitation and Relative Quantitation assays can be run fast. Allelic Discrimination (SNP) assays and plus/minus assays using internal positive controls cannot be run fast.How stable is an experiment set up with the PerfeCTa qPCR Fastmix Kit at room temperature?For optimal results, it is recommended to directly run the reaction plate after setting up the reaction. If for any reason a reaction plate cannot be run within 2 hours of completing the reaction setup, then freeze/refrigerate the reaction plate until it can be loaded and run on a Real-Time PCR System.CloningIs there a preferred method for transforming E.coli with the assembled DNA?The products of the repliQa HiFi Assembly reaction can be introduced into E.coli either by chemical transformation or electroporation. If electroporation is to be used for transforming cells, we recommend first diluting the assembly reaction 1:5 in high purity water.What incubation temperature should be used for the assembly reaction?The repliQa HiFi Assembly Mix is optimized for assembly at 50°C. We do not recommend performing the reactions below 48°C or above 52°C.What are the advantages of the repliQa HiFi Assembly Mix for cloning?The repliQa HiFi Assembly Mix allows simultaneous cloning of multiple insert fragments into vectors without creating scars. In contrast to traditional cloning, there is no requirement for restriction endonucleases and unique restriction sites.Is it necessary to purify the PCR fragments that will be used in the assembly reaction?Purification of PCR product using a spin column-based cleanup or other method is not required but is highly recommended to achieve highest efficiency fragment assembly.What is the recommended amount of sequence overlap?The repliQa HiFi Assembly Mix is optimized for assembly of fragments with overlap regions between 15 – 60 bp.Is an E.coli strain supplied with the repliQa mix?The repliQa HiFi Assembly Mix does not include competent E.coli. Most common E.coli cloning strains are compatible with the kit and are available from numerous commercial sources.What E.coli strains are compatible with the repliQa assembly mix?Common E.coli cloning strains and derivatives are compatible with the repliQa HiFi Assembly Mix.At what concentration is the repliQa HiFi Assembly Mix supplied?The repliQa HiFi Assembly Mix comes with a 10X reaction buffer containing dNTPs, magnesium, and cofactors. This 10X concentration allows for use of higher volumes of nucleic acids and flexibility in design of assembly reactions compared to alternative kits or approaches.How is the pmol input of DNA fragment amounts calculated?Calculate the number of pmols of each fragment using the following formula: pmoles = (weight in ng) x 1000/(bp x 662).How many fragments can be assembled in a single reaction?Quantabio routinely generates assemblies of up to six DNA fragments, though the repliQa HiFi Assembly Mix has been successfully used for more complex assemblies.When should the DpnI be used?If plasmid DNA is used as PCR template for generation of fragments for assembly, it is recommended to treat the PCR reactions with DpnI prior to the assembly reaction to eliminate residual methylated plasmid that could give rise to colonies lacking the assembled product of interest.Can the reaction time be reduced to 15 minutes rather than 1 hour?For reactions using three or fewer fragments, the incubation time can be shortened to 15 minutes if necessary.Why are plots of the number of colonies important?The number of colonies is indicative of transformation efficiency, which in turn is indicative of reaction assembly efficiency. The high efficiency, reliability, and consistency of the repliQa HiFi Assembly Mix improves the likelihood of obtaining correct assemblies in situations where alternative kits or approaches fail.What are the minimum and maximum fragment sizes that can be assembled?Quantabio has effectively assembled fragments down to 116 bp (70 bp insert and two 23 bp overlaps). When attempting to clone small fragments, we recommend using 2- to 5-fold molar excess of fragments. Regarding maximum sizes, there should be no limit on what can be assembled. The only limitation is from the challenge of pipetting large assemblies without causing damage or shearing.
microRNA ProfilingHow does the qScript microRNA Quantification System distinguish mature microRNAs from precursor-microRNA (pre-miRNA) hairpin structures?The qScript microRNA Quantification System is specific for mature microRNAs. The PerfeCTa microRNA Assays primarily distinguish mature microRNAs from pre-microRNAs based on proprietary design features of the PerfeCTa microRNA assay primer. Efficacy of pre-miRNA as template also depends on how well the pre-miRNA can be poly(A) tailed. Recessed and blunt 3’-ends of pre-miRNA hairpins are a poor substrate for poly(A) polymerase tailing. In addition, the hairpin structure of pre-miRNAs may not be efficiently amplified during PCR due to the stability of the secondary structure of the template competing with the annealing of the PCR primer. Finally, if a pre-miRNA product is amplified it will typically be larger in size than the mature microRNA product and thus distinguished by melting at a higher temperature.Can the qScript microRNA Quantification System be used to quantify mRNA as well as miRNA?Although the kit has been designed to work optimally for microRNA detection, mRNA will be represented in the microRNA cDNA population. The representation of mRNA, however, will favor amplicons designed near the 3’-end of transcripts since the reverse transcriptase reaction is primed using an oligo-dT adapter primer. Equal representation of the entire length of mRNA transcripts would require a random priming method.Can the qScript microRNA system be used with serum and plasma samples?The qScript microRNA Quantification System can be used with serum and plasma samples. Quanta participated in the miRQC study where the microRNA kit performed very well in every category with each sample including the most challenging serum samples. Serum/plasma RNAs can be purified using TRIzol, TRIzol LS or Qiagen kits (either miRNeasy Mini or miRNeasy Serum/Plasma). Since the RNA concentrations are low in serum and plasma samples, it is important to include a co-precipitant like glycogen or MS2 phage RNA at the RNA precipitation step. There are numerous references on the topic in the literature (see whitepaper from Qiagen). We would recommend using a synthetic RNA oligo spike-in control. We offer 4 different C. elegans microRNA assays that can be used for this purpose. You must first order a custom RNA oligonucleotide synthesis for one of the C. elegans microRNAs. Samples are spiked with a dilution of the synthetic RNA oligo template immediately following lysis and homogenization of the sample. Add enough synthetic RNA oligo to each sample so that there will be at least 10,000 copies per well in each qPCR reaction. For example if 1/10 of the RNA prep was used to make cDNA and 1/100 of the cDNA was used in each qPCR then spike the sample with 107 copies of the synthetic RNA oligo. Use equal volumes of sample in the cDNA synthesis and qPCR reactions since RNA concentration cannot be accurately determined. The qPCR results of the C. elegans assay can then be used to normalize for the efficiency of the RNA sample preparation method. Since the typical small RNA controls are usually not detectable in serum and plasma samples you must select a panel of microRNA assays that are known to be detectable in serum. Following some preliminary experimentation, choose a subset of assays whose levels are determined to be invariant with respect to the biological model system of interest (for example normal and disease tissue) to use as data normalization controls.What is the amount of cDNA used per TaqMan or SYBR qPCR Assay?Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 100 pg to 100 ng genomic DNA. For more information, please consult the PerfeCta qPCR Supermixes or PerfeCta SYBR Green Supermixes protocols.Is there a SYBR® Green Fast Master Mix currently availableYes. PerfeCTa SYBR Green Supermix is available. For further information, please consult the Kit protocol or product information.The microRNA kit arrived thawed without dry ice, will the kit still work?We have tested the kit after 1 day and 3 days at room temperature and the results were identical to material stored at -20 C. The functionality of the kit components can be tested using dilutions of synthetic microRNA oligonucleotides. In addition, the Human Control Primer (included in the qScript microRNA cDNA Synthesis Kit) can be used as a template for cDNA synthesis followed by qPCR. Start with at least a 1/1000 dilution of the human control primer and then make 3 or more 1/10 dilutions. Use 5 uL of each dilution in 10 uL reactions with and without the poly(A) polymerase (substituting water for enzyme in the no-poly(A) polymerase reactions). Run the reverse transcriptase reactions for each and dilute the cDNAs to 100 uL by adding 80 uL of TE0.1 (10 mM Tris-HCl, pH 8, 0.1 mM EDTA) or water. Run 1 to 5 uL of the diluted cDNA reactions in 25 uL qPCR reactions using the human control assay primer and universal PCR primer. You should be able to generate a standard curve and see significant differences in Ct values between the samples prepared with and without poly(A) polymerase.What RNA isolation kit or protocols are compatible with the qScript microRNA Quantification System?Any RNA isolation kit or protocol that retains or enriches small RNAs is compatible. RNA isolation kits that enrich mRNA and exclude small RNAs are not compatible. Please refer to the user manual of your RNA isolation kit to be sure that it retains and does not exclude small RNAs. We have had good success with Trizol prepared total RNA. Also with cell lines you can use a direct cell lysis reagent (RNAGEM from ZyGEM) eliminating the RNA preparation step. We do not recommend using RNA preparations that are enriched for small RNAs because they can introduce a bias between different sample preparations.
Miscellaneous QuestionsWhat 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.Is MgCl included in the Max Capacity cDNA Kit ?Magnesium Chloride is contained within the 5X Max Capacity cDNA Kit. For more information please consult the Max Capacity cDNA Kit protocol.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°CDo 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 temperatureWhat 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 UDGWhat 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-PCROne-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 mixThe 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 & StabilityMMLV 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 activityWhat 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 protocolsMw and Size of Taq PolymeraseTaq DNA Polymerase is an 832 amino acid, single subunit enzyme with a MW of 94,000.Optimal pH for Taq polymeraseTaq polymerase is active from pH 7.5-9.5. The unit assay is performed at pH 9.3 in TAPs buffer.Extension rate of TaqTaq 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 many reactions can I perform with the High Capacity cDNA Kit?The Max-Capacity cDNA Kit contains sufficient reagent quantities to perform x reverse transcription reactions each with a reaction size of 50µl. For further information, please consult the High-Capacity cDNA Kit protocol or product information.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 qPCRsWhat 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 polymeraseThe 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 bufferThe 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 ChlorideUnits of AccuStart Taq DNA polymerase to be used in PCR1-1.5 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 polymeraseTaq 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 activityTaq 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 TaqAccuStart 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 PolymeraseOne 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, 95CTest Primer DesignTest Answer