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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 qPCRs