Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. In order to minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

• Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
• Use only fresh PCR-grade reagents and disposable lab ware.
• Treat any lab ware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
• Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, cloning, etc.
• Change the lab bench pads/papers often and decontaminate lab ware (racks, pipettors, etc.) before each use by washing lab benches and lab ware with 10% bleach, and/or exposing them to UV light for at least 10 minutes. This serves to degrade and/or inactivate any contaminating DNA.
• Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.

The most important prerequisite for any gene expression analysis experiment is the preparation of consistent, high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment. Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It also is a source of false positive signals in RT-PCR experiments. RNase contamination degrades your RNA samples, causes low signal and false negative results in the PCR. Residual polysaccharides, collagen, other macromolecules or organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

Ribonucleases are the #1 threat to any RNA isolation procedure. In addition, co-purification of inhibitory contaminants is a major problem when isolating RNA from certain tissue sources. In order to minimize the threat, gloves should be worn at all times, and special care must be taken to use RNase-free reagents and lab ware. In addition tissue/cell lysis steps are typically carried out with lysis buffers containing guanidine isothiocyanate, a potent protein denaturant. It is very important to use a sufficient amount of lysis buffer during RNA isolation. We recommend using at least 10 X volume of lysis buffer vs tissue/cell pellet. It is more challenging to isolate high quality RNA from tissue samples than from cultured cells, especially those tissues containing high levels of RNase, or difficult-to-homogenize tissues. Examples of such tissues include liver, heart, skin and conjunctive tissues. Many tissue samples also contain difficult-to-remove contaminants (such as polysaccharides, collagen, fats, lipids or fibrous components) that may interfere with subsequent enzymatic reactions if not removed from the RNA preparation.

We recommend the use of QIAGEN RNeasy Mini Kits. Cultured cells, and freshly isolated white blood cells, may be harvested by centrifugation, and used directly with this kit. For the isolation of high quality RNA from animal tissues, we recommend the QIAGEN RNeasy Lipid Tissue Mini Kits.

The chemical cross-links present in FFPE blocks or slides complicate RNA purification, and also inhibit downstream enzymatic reactions. If RNA prepared from such samples is contaminated with inhibitory chemicals, the sensitivity of subsequent gene expression analyses (such as qRT-PCR) will be dramatically reduced. In order to maximize both the quality and the yield of the RNA prepared from such samples, it is imperative that a procedure be used that efficiently reverses the cross-links and completely dissolves the tissue.

For best results, all RNA samples should be suspended in RNase-free water. Alternatively, RNase-free 1 mM sodium citrate (pH 6.5) or 10mM Tris buffer (pH 7.0) may be used. DO NOT use DEPC-treated water, as most DEPC preparations are contaminated with molecules that are inhibitory to reverse transcription and/or PCR. For long term storage, RNA preps may be stored at -70 ºC in RNase-free water, or the buffers listed above, or precipitated in ethanol or isopropanol. In order to avoid repeated freeze-thaw cycles, it is recommended that frozen RNA samples be stored as multiple, single-use aliquots.

All RNA samples should be assessed spectrophotometrically (diluted in 10mM Tris, pH 8.0), and electrophoretically, and should meet the following specifications:

• Total RNA concentration by A₂₆₀ should be greater than 40 µg/ml
• A₂₆₀: A₂₈₀ ratio should be 1.8 to 2.0
• A₂₆₀: A₂₃₀ ratio should be greater than 1.7
• Analysis of ~100ng of total RNA on an Agilent Bioanalyzer using an RNA 6000 Nano LabChip, or analysis of 1.5 μg of total RNA on a denaturing 2.0% agarose gel containing ethidium bromide (0.5 μg/ml) should contain sharp 28S and 18S rRNA bands, with no smearing at their low molecular weight edge. The 28S:18S band intensity ratio should be ~2:1. When utilizing the RNA 6000 Nano LabChip for RNA analysis, the RNA should have a RIN (RNA integrity) score of 7.0 or higher.

In addition to the above quality control tests, one can also use the SABiosciences RT² RNA QC PCR Array for human (PAHS-999), mouse (PAMM-999), or rat (PARN-999). These arrays allow the rapid assessment of high and low housekeeping gene expression levels; reverse transcription and polymerase chain reaction efficiency; and genomic and general DNA contamination.

If you suspect that your RNA preparation contains RNase contamination, re-purify the preparation over a spin-column based method.

Carry out all procedures in a "DNA-free" workspace (see the first Sample Preparation FAQ, above). Be sure to include any DNase treatment steps in the recommended RNA isolation procedure or treat RNA separately with RNase-free DNase followed by re-purification using a spin-column based method. Be sure to double both the units of enzyme and the incubation time recommended by the RNase-free DNase manufacturer. In order to minimize DNA contamination in your RNA preparations, and avoid the need for supplemental DNase treatments, we recommend using the SABiosciences RT² First Strand Kit, which includes a highly efficient genomic DNA elimination step before reverse transcription.

Pooling RNA from different sources should only be done when there is not enough sample. We recommend running biological replicates.

There is no need to run a standard curve before doing an actual experiment. Usually we recommend starting with 1000ng of total RNA for a 96 well PCR Array.

Carry out all procedures in a "DNA-free" workspace (see the first Sample Preparation FAQ, above). Be sure to include any DNase treatment steps in the recommended RNA isolation procedure or treat RNA separately with RNase-free DNase followed by re-purification using a spin-column based method. Be sure to double both the units of enzyme and the incubation time recommended by the RNase-free DNase manufacturer. In order to minimize DNA contamination in your RNA preparations, and avoid the need for supplemental DNase treatments, we recommend using the SABiosciences' RT² First Strand Kit (C-03), which includes a highly efficient genomic DNA elimination step before reverse transcription. NOTE: Our Chemistries are NOT COMPATIBLE with AMBION's Turbo DNA-free Kits.

Three options exist for priming a reverse transcription reaction. (1) The classic approach is to utilize oligo(dT) primers. These primers are typically ~20 bases in length, and anneal to the polyA tails of mRNA. By targeting the mRNA fraction, the complexity of the resultant cDNA population is dramatically reduced, since rRNA and tRNA species will not serve as templates in the reaction. The drawback of using oligo(dT) primers is that the resultant cDNA population will have a 3'bias, thus compromising the effectiveness of PCR primers targeting the 5'ends of transcripts. In addition, due to the 3'bias, fragmented samples lacking a polyA tail will not be reverse transcribed. (2) Utilizing random primers is another popular strategy for priming reverse transcription. These are a random mixture of the four DNA bases of a specified oligo length. Random hexamer mixes are commonly used, consisting of 4096 sequences (4⁶). Each of these primers will anneal anywhere the complementary sequence exists within a given RNA molecule (including rRNA, tRNA, mRNA, and any fragments of these species). Reverse transcription using random primers overcomes the worries of RNA 2º structure, and RNA fragments, which are common headaches when using oligo(dT) primers. Many researchers utilize blends of oligo(dT) and random primers, in order to maximize cDNA yields. If one intends to utilize 18S rRNA as an internal normalizer in gene expression studies, a random priming strategy must be used, since rRNA do not carry a polyA tail. (3) When studying a single gene that expresses at very low levels, researchers will sometimes utilize gene-specific primers, resulting in the production of a cDNA population of minimal complexity. Obviously, such cDNA preparations can not be utilized for multigenic gene expression studies.

Traditional two-step RT-PCR methods perform the reverse transcription (RT) reaction and the polymerase chain reaction (PCR) sequentially in separate tubes with separate buffers optimized for each step. One-step RT-PCR methods, combine both the RT reaction and the PCR. A single buffer is optimized for both reactions, and the reactions occur in the same tube. The key benefits of a one-step approach are that they are more time-efficient, and reduce the risk of contamination by extraneous DNA. The disadvantage of one-step methods is that the fidelity of both the reverse transcriptase and the DNA polymerase enzymes can be reduced because the common buffer optimized to use the two enzymes in the same tube can actually be sub-optimal relative to the buffers specifically optimized for each individual enzyme.

The most rigorous method to detect genomic DNA contamination is to perform a No Reverse Transcriptase (NRT) control. The PCR will have no cDNA template derived from mRNA, and any detectable product could only have been derived from genomic DNA contamination.

Quantitative PCR, also referred to as qPCR or real-time PCR, is a reaction from which the accumulation of the amplicon product can be monitored in real-time as the polymerase chain reaction proceeds, usually as a fluorescence intensity. The fluorescent signal generated during the exponential phase of the PCR reaction is directly proportional to the amount of amplicon produced, and is therefore a powerful method for quantitating the amount of template DNA used in the reaction. Compared to end-point PCR approaches, qPCR provides superior sensitivity, dynamic range, and precision, while eliminating the need for post-PCR sample processing.

The types of probes utilized for qPCR fall into two classes, those that are not sequence-specific, and those that are sequence-specific. The most popular non-sequence-specific probe chemistry is SYBR Green, which binds to the minor groove of double-stranded DNA, and fluoresces 1000-fold more efficiently when bound, than when free in solution. It is the most cost-effective, and convenient chemistry for qPCR. Historically, researchers have worried that SYBR Green chemistry would show inferior specificity, when compared to the sequence-specific probe chemistries. SABiosciences has eliminated that concern, through the development of complementary SYBR Green-based RT² qPCR Master Mixes and RT² qPCR Primer Assays, which are available for any gene in the human, mouse, or rat genome. The sequence-specific qPCR probe chemistries fall into two classes. The bimolecular probes consist of a dual-labeled probe used in combination with flanking forward and reverse primers. The TaqMan and Molecular Beacons probes are popular examples of these chemistries. The unimolecular sequence-specific probes consist of a dual-labeled hairpin probe which is covalently linked to a forward primer, and is used in combination with an unlabeled reverse primer. The Amplifluor and Scorpion probes are popular examples of this class.

There are several manufacturers of high quality qPCR/real-time thermal cycling instruments. These include QIAGEN, ABI, BioRad, Stratagene, Eppendorf, Roche, and Cepheid. The important thing to keep in mind is that, once you select an instrument to use, you use compatible 96 or 384 well plates, and qPCR Master Mixes that are optimized for use in that particular instrument.

The ROX and Fluorescein dyes are used in select qPCR instruments to normalize their optics for most fluorescent fluctuations, to compensate for well-to-well volume variations, to regulate minor volume differences and changes in concentration, and to optimize detection precision.

The three most common negative controls included in a qPCR and/or qRT-PCR experiment are as follows:
1. No Template Control (NTC) omits any DNA or RNA template from a reaction, and serves as a general control for extraneous nucleic acid contamination. When using SYBR Green chemistry, this also serves as an important control for primer dimer formation.
2. No Reverse Transcriptase Control (NRT) or Minus Reverse Transcriptase Control (MRT) involves carrying out the reverse transcription step of a qRT-PCR experiment in the absence of reverse transcriptase. This control assesses the amount of DNA contamination present in an RNA preparation.
3. No Amplification Control (NAC) omits the DNA polymerase from the PCR reaction. This is a control for background fluorescence that is not a function of the PCR. Such fluorescence is typically attributable to the use of a degraded dual-labeled probe. This control is unnecessary when utilizing SYBR-Green probe chemistries.

It is critical to include appropriate positive controls in a qPCR experiment, in order to determine if false negatives are being detected in the experiment. Positive controls fall into one of two classes.
1. Exogenous positive controls refer to the use of external DNA or RNA carrying a target of interest. If these positive controls are assayed in separate wells/tubes from the experimental sample, they serve as a control for whether or not the reverse transcription and/or PCR reaction conditions are optimal. Additionally, the exogenous DNA or RNA positive controls may be spiked into the experimental sample(s), and assayed in parallel to, or in a multiplex format with, the target of interest. These control reactions assess if the samples contain any components that inhibit reverse transcription and/or PCR.
2. Endogenous positive controls refer to the use of a native target that is present in the experimental sample(s) of interest, but is different from the target under study. These types of controls are often referred to as normalizers, and are typically utilized to correct for quantity and quality differences between samples.

If you are unsure of the correct housekeeping gene(s), review the literature in your field to determine which gene(s) other researchers like yourself commonly use. It is recommended that multiple housekeeping genes be utilized for each gene expression experiment, in order to account for any impact that an experimental condition may have on the expression of an individual housekeeping gene. For a systematic assessment of which housekeeping genes are appropriate for your specific experimental conditions, we recommend using the SABiosciences Housekeeping Genes PCR Arrays for human (PAHS-000), mouse (PAMM-000), or rat (PARN-000). These arrays consist of 8 sets of 12 common housekeeping genes. They are a valuable tool for easily identifying genes with a constant level of expression among your different experimental conditions.

Because of its invariant expression across tissues, cells and experimental treatments, 18S ribosomal RNA is a widely used control for most qRT-PCR analyses. However, due to its extremely high expression in most cell types, it can sometimes be challenging to use 18S rRNA as an endogenous normalizer for other gene expression assays in the same reaction.

HotStart PCR is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. Lack of sensitivity or specificity is most often caused by the amplification of non-specific priming events, such as primer dimers, that usually occur at the lower temperatures where reactions are set up. Although the thermostable DNA-dependent DNA polymerases have optimal activity at higher temperatures, they do also have some activity at lower temperature where they may amplify these non-specific priming events. HotStart enzymes are inactive at room temperature, and require heating at nucleic acid melting temperatures in order to be activated. In this way, the non-specific priming events are melted before the enzyme can amplify them. During the PCR cycles, the temperature never drops low enough during the annealing of the gene-specific primers for the non-specific priming events to re-occur, resulting exclusively in amplification of the target of interest. When using a HotStart DNA polymerase, it is critical that the initial denaturation step in the experiment be of sufficient duration to fully activate the enzyme.

Primer dimers are undesirable side-products of PCR and result when one primer anneals to another primer, forming a substrate for amplification by the DNA polymerase during PCR. This secondary product contributes to the SYBR Green qPCR signal for a gene, artificially increasing its apparent expression level. In end-point PCR, the amplification of the primer dimer can decrease the level of PCR reagents needed for the amplification of the gene-specific product of interest, thereby artificially decreasing its apparent expression level. Rigorous optimization of qPCR Primer Assays and qPCR master mixes can lead to the development of optimal assay conditions, which virtually eliminate the production of primer dimers.

Dissociation curves are carried out at the end of a PCR experiment by following a three step procedure First, all of the components are denatured at 95C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve. By analyzing the first derivative of such a curve, one can readily assess the homogeneity of the PCR products, including the presence of primer dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry, due to this reagent's lack of sequence specificity.

Be sure to carefully and completely seal the qPCR assay plate with fresh optical thin-wall 8-cap strips or adhesive optical film before the plate is placed into your thermal cycler. In addition, refer to your instrument's user's manual to determine if the thermal cycler manufacturer recommends the use of a plate compression pad during the run.

There are less than a dozen genes encoded by the mitochondrial genome (all other mitochondrial proteins are encoded by nuclear genes), and they are all transcribed as one transcript (just like any prokaryote), so distinguishing the expression of individual genes by real-time RT-PCR is quite impossible.

The Rn value, or normalized reporter value, is the fluorescent signal from SYBR Green normalized to (divided by) the signal of the passive reference dye for a given reaction. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter reliably calculates the magnitude of the specific signal generated from a given set of PCR conditions. For more information, please refer to your instrument's user manual.

The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.

Make a note of those wells that show signs of evaporation, in order to qualify the data calculated for those wells. To insure that evaporation does not occur in the future, be sure to carefully and completely seal the qPCR assay plate with fresh optical thin-wall 8-cap strips or adhesive optical film before the plate is placed into your thermal cycler. In addition, refer to your instrument's user's manual to determine if the thermal cycler manufacturer recommends the use of a compression pad during the run.

You may be using too much template. Use less input total RNA for reverse transcription, or use template at a greater dilution factor (lower concentration). Do not pipet a volume of less than 1 μl.

There are several reasons for not seeing a PCR product. 1. The corresponding gene may not be expressed above the limit of detection of the qRT-PCR assay method. 2. There may have been experimental error, in which case, use a template known to contain the gene of interest as a positive control to troubleshoot the PCR reagents and experimental procedure. 3. The RNA may have been of poor quality, in which case, be sure to perform all of the recommended quality control checks on the RNA sample (see Sample Preparation FAQs, above). 4. There may not have been enough template, in which case, use more input total RNA, or use the template at a lower dilution factor (higher concentration), or use a larger volume of template. 5. Another possible explanation pertains to when one is trying to detect cellular expression from an exogenous vector that has been introduced into a cell. If the vector expresses only the open reading frame (ORF) of the gene of interest, and the qPCR primers being used amplify a target within the 5' or 3' UTR (untranslated region) of the gene, the transcript will not be detected.

There is DNA contamination somewhere in your PCR assay system. Use only PCR-grade reagents and lab ware. Wear gloves throughout the procedure. Always use fresh pipette tips, water and other reagents. Do not leave lab ware (open tubes and tip boxes) exposed to the air for long periods of time. The most common source of DNA contamination comes from the PCR products of previous experiments. Avoid the spread of any PCR products into the air of your working environment. Close all tubes containing PCR products once you are finished adding or removing volumes. Discard all tips or tubes that have been in contact with PCR products into a container of bleach. Clean your bench and your pipettors often. Some researchers expose lab ware with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers.

The most rigorous method to detect genomic DNA contamination, particularly with the RT² PCR Primer Assays, is to perform a No Reverse Transcriptase (NRT) control. The PCR will have no cDNA template derived from mRNA, and any detectable product could only have been derived from genomic DNA contamination.

Assuming 100% amplification efficiency, each step increase in Ct value represents a doubling in the amount of qPCR template. Therefore, evaluating the difference in Ct values between the qPCR assay, and its matching NRT control, leads to the following predictions:

CtNRT - Ct+RT	Fraction of gene expression signal due to contaminating DNA	Percentage of gene expression signal due to contaminating DNA
1	(1/21) = 1/2	50%
2	(1/22) = 1/4	25%
3	(1/23) = 1/8	13%
4	(1/24) = 1/16	6%
5	(1/25) = 1/32	3%

The optics of the qPCR instrumentation may be saturating due to improper instrument settings. Please consult with your instrument manufacturer for more details.

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings. Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

There are several reasons for seeing multiple peaks in the real-time dissociation curve. Always verify amplicon production by agarose gel electrophoresis. Larger bands could be due to genomic DNA contamination, which can be verified using a No Reverse Transcription (NRT) control. Smaller bands could be due to the presence of primer dimers. Extra bands could also be due to the presence of un-annotated alternative transcripts or splice variants in your total RNA sample.

When using the standard curve method, the quantity of each experimental sample is first determined using a standard curve, and is then expressed relative to a calibrator sample. In order to use this quantification method, prepare five (5) 2-fold, 5-fold, or 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values. In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. Confirm that the correlation coefficient (R2) for the line is 0.99 or greater. This plot is then used as a standard or calibration curve for extrapolating relative expression level information for the same gene of interest in unknown experimental samples. The relative quantification calibration curve result for the gene of interest is normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to get a fold change in expression. A standard or calibration curve must be generated separately for each gene of interest and each housekeeping gene.

Absolute Quantification determines expression levels in absolute numbers of copies. Relative Quantification determines fold changes in expression between two samples. In absolute quantification, the precise amount of the message or template used for the curve is known. In relative quantification, the template is simply known to contain the message of interest in high abundance, but its absolute amount is not necessarily known. Unknowns are compared to either standard curve and a value is extrapolated. The absolute quantification standard curve provides the final answer. The relative quantification calibration curve result for the gene of interest is normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to obtain a fold change.

In the comparative or ΔΔCt method of qPCR data analysis, the Ct values obtained from two different experimental RNA samples are directly normalized to a housekeeping gene and then compared. This method assumes that the amplification efficiencies of the gene of interest and the housekeeping genes are close to 100 percent (meaning a standard or calibration curve slope of -3.32) First, the difference between the Ct values (ΔCt) of the gene of interest and the housekeeping gene is calculated for each experimental sample. Then, the difference in the ΔCt values between the experimental and control samples ΔΔCt is calculated. The fold-change in expression of the gene of interest between the two samples is then equal to 2^(-ΔΔCt).

Prepare five (5) 2-fold, 5-fold, or 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values. In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. Confirm that the correlation coefficient (R2) is 0.99 or greater. The closer the slope of this straight line is to -3.32, the closer the amplification efficiency is to 100 percent.

The amplification efficiency = [10^(-1/slope)] - 1

Alternatively, a number of data analysis models have been developed that enable the calculation of PCR amplification efficiencies from individual amplification plots, without the use of standard curves. These include the Data Analysis for Real-time PCR (DART-PCR), LinReg, and the Real-time PCR Miner algorithms. Because these methods do not require the generation of standard curves, they are well suited for large scale experiments

The template that you chose to use in generating your standard curve may not express your gene of interest abundantly enough to be detected after the several 10-fold serial dilutions required for the standard curve. In such a case, many of the standard curve reactions should be yielding high Ct values (> 30). You can lower the serial dilution factor to 2-fold, and generate a new standard curve. You can also try using an alternate source of template for the standard curve reactions, such as cDNA derived from a universal source of RNA, cDNA derived from a full-length in vitro transcript of the gene of interest, or even a full-length cDNA clone of the gene of interest.

Prepare five (5) 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values. In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. The linear range of this plot is the linear dynamic range of the qPCR assay.

Replication of a gene on the PCR Array is necessary to know the technical reproducibility of that PCR assay. We have 2 assays to monitor this technical reproducibility, the RTC and PPC assays. Based on our experiences, most of the variability in gene expression analysis when using qPCR is at the level of the biological sample and not associated with the individual PCR step. If you would like to test this for yourself, you can use the same RNA sample and do three separate RT reactions and load each sample into its own PCR Array (this will test the technical reproducibility from the RT step forward) or start with the same cDNA sample and load three PCR Arrays (this will test the technical reproducibility from the PCR step forward) and compare the final fold change values.

Certainly. All you need to do is to organize your data into a “custom PCR Array” file. When you upload it to the website, use the custom PCR array name CUSTOM. The locations of the blank excel spreadsheet is: http://www.sabiosciences.com/pcrarraydataanalysis.php#custom

The fold difference (fold change) is calculated by the equation 2(-??C(t)). For the fold regulation, we take any fold regulation (or fold change) numbers less than 1 (meaning that the gene is down regulated) and take the negative inverse, changing the fractional number into a whole number. The values of the numbers are the same, we have only changed the representation of the value. For example if ABL1 has a fold change value of 0.31, this is equivalent ABL1 having a fold regulation of -3.2 fold. (ABL1 is down regulated by 3.2 fold).

Your data needs to be arranged so that the Raw C(t) or C(p) value corresponds to the correct well location on the PCR Array. A blank spreadsheet is available at: http://www.sabiosciences.com/pcr/template.xls

If you are starting with a 384 well plate which is in the 4x96 layout, then you will need to deconvolve the data using the :384-Well Format E/G Data Analysis Patch tool located at http://www.sabiosciences.com/pcrarraydataanalysis.php

The C(t) value is where the raw florescent traces cross the threshold line. To set your Ct, use your real-time PCR machine and click on auto baseline. Then look at your raw florescent traces in the log view. Move the threshold line somewhere in the lower 1/3 of the florescent traces. The PPC assays (wells H10,H11,H12) should be between 18 and 22.

The significance for the Fold Change is dependent on what the final use of your data is going to be. If you have run three biological replicates, and there are no significant fold changes between you treated and control, then that particular gene is probably not changing. I would not throw out the data, but report it as a gene that is not changing. You can estimate the number of samples that will need to be run based on your fold change, variance and p values using a power test.

It means that there was no Ct value calculated for that gene.

The set Ct cutoff is the value that empty, or wells without Ct values (such as undetermined or N/A) will be set to for data analysis. Additionally, any Ct values above the set Ct cutoff will be set to that value. This allows us to calculate “artificial” Fold Change calculations for genes that are not expressed versus samples where the gene is expressed. Care needs to be taken when looking at the fold change values where samples have Cts greater than the set Ct cutoff, because the fold change value will be artificial.

You do not need to do anything with the undetermined wells? Undetermined wells will automatically be changed to the “Set Ct Cut-off” value during data analysis (35 by default)

We determine the amplification efficiency during wet bench testing of our assays using standard curve dilutions, or by single curve analysis. If you would like to calculate the efficiency of each curve using single curve analysis, then you can try Real-Time PCR Miner, LinReg or Dart PCR. Each of these can be found using a GOOGLE search.

You can manually set the threshold line. If you are using a catalogued PCR Array, the PPC values should be 20 +/- 2 Cts. Use the same threshold on all of your PCR Arrays.

No. Data Analysis can be done with a little as 2 PCR Arrays. Whether or not you run a sample in triplicate is determined by experimental setup and what you are going to use the data for.

Each PCR Array has 5 housekeeping genes. You can use one or an average of the most stable ones to do data analysis.

The best way to set the threshold is to make sure that your PPC values are between 18 and 22. I would look my first PCR Array, set it so that the PPC is at 20, and see if the same threshold fits for the rest of the arrays.

Please log in to our website, or first register with us if you never have before. Under the "Support" tab in the top menu bar, click "User Manual". You may then download any User Manual that you need.

You may call 1-888-503-3187 for technical support, or click the active link "Email a Question/Suggestion" from any products page on our website to file a case, or send an email to support@sabiosciences.com. Technical Support representatives are generally available Monday to Friday from 9 AM to 6 PM EST.

The performance of our RT² qPCR Primer Assays have been tested, and are guaranteed with, our RT² SYBR Green qPCR master mixes only. Our primer design algorithm accounts for our master mix buffer system parameters such as ionic strength and magnesium chloride concentration. We have not tested our Primer Assays with other sources of master mix and their buffer system parameters. We only guarantee that our primers will perform optimally with our master mixes. We do not guarantee optimal performance with other sources of master mix, without requiring further optimization studies by the end-user.

You need: 1. A SABiosciences RT² SYBR Green qPCR master mix that matches the qPCR instrument in your laboratory; 2. RT² qPCR Primer Assays for your target genes; 3. A Housekeeping gene RT² qPCR Primer Assay. We also recommend using our RT² First Strand cDNA Synthesis Kit for reverse transcription.

RT² qPCR Primer Assays are available for any gene in the human, mouse, or rat genome. In addition, we also support custom primer designs for other species. You may call our Technical Support Team at 1-888-503-3187, in order to receive a quote for the design and manufacture of custom primers.

On the product information sheet, we include the reference position for the gene-specific amplicon relative to the RT² qPCR Primer Assay's corresponding RefSeq number. Journals accept the catalog number and the reference position for publication purposes. Customers may also use the reference position, the RefSeq number, and the NCBI database to insure that the amplicons are indeed gene-specific and even determine the approximate region amplified by the primers.

For SYBR Green-based qPCR detection, the most important parameter for primer design is the generation of only a single gene-specific amplicon with high amplification efficiency, without the production of primer dimers. Primer assays amplifying short products contained within a single exon meet this parameter most optimally. Primers that cross exon-intron junctions may still detect processed pseudogenes, heteronuclear RNA (hnRNA), as well as unannotated alternative transcripts and splice variants, thus complicating SYBR Green-based qPCR detection.

Each RT² PCR Primer Assay is validated at SABiosciences, with both a real-time and conventional PCR quality control assay. These assays are carried out using a single source of genomic DNA. In order to pass QC, each primer assay must generate a single band of the correct predicted size by agarose gel electrophoresis and a single peak in the real-time dissociation curve without the appearance of primer dimers. The amplification efficiencies (DART method) and sensitivities of each primer set are also validated.

When a RT² PCR Primer Assay™ recognizes another transcript of the same gene, the resulting signal by real-time or end-point detection represents the sum of the relative expression of all of the transcripts detected by the Primer Assay. When alternative transcripts or splice variants are known and annotated in the public databases, the RT² PCR Primer Assay are designed to generate an amplicon in common to as many of those known transcripts as possible

The performance of our RT² qPCR Primer Assays have been tested, and are guaranteed with, our RT² SYBR Green qPCR master mixes only. Different master mixes have been optimized and are available for different qPCR instrumentation, because each instrument uses a different reference dye to normalize their optics. In order to guarantee that your RT² qPCR Primer Assays will work right the first time in your hands, we need to make sure that you receive the correct RT² SYBR Green qPCR master mix for your real-time instrument.

Our RT² qPCR Primer Assays may be used on any real-time instrument. SABiosciences offers qPCR solutions for the most popular qPCR instrumentation, including those from QIAGEN, ABI, BioRad, Stratagene. SABiosciences has written instrument-specific protocols for select instruments, which can be accessed at the following link:
http://www.sabiosciences.com/pcrarrayprotocolfiles.php

We can only guarantee the performance of RT² qPCR Primer Assays with our RT² qPCR Master mixes. Our master mix components and primer design algorithm were optimized together to guarantee production of single bands of the predicted size. When we do test other sources of master mix with our Primer Assays, we frequently see primer dimers and other non-specific products that confound SYBR-Green based qPCR detection.

The useful range of input total RNA for the first strand cDNA template synthesis (reverse transcription) reaction is between 100ng and 5 µg. For initial experiments, we recommend using between 0.5 to 1 µg of input total RNA, and using 1 µl of either undiluted template or template pre-diluted 1:10 for each 25-µl RT² qPCR Assay reaction.

Yes, an RT² qPCR Primer Assay corresponding to each gene on every one of SABiosciences' microarray products, or any other microarray product, is available.

The sudden failure of a re-used RT² qPCR master mix is most likely due to repeated warming of the same 200-reaction scale product. We do not recommend repeatedly removing the same vial of PCR master mix from the -20 ºC freezer. The enzyme activity will decrease over time, under these conditions. Instead, upon receiving the master mix, divide into aliquots of a volume that you predict you will use for each day's experiment. Any unused portion of an aliquot should either be discarded or saved, noting that it has been used previously, for less critical uses or experiments. Make sure that you do not store the RT² qPCR master mixes frozen at -80 ºC, as this will kill master mix activity.

For detailed pricing information on any of our PCR products, please visit the following web address: http://www.sabiosciences.com/price.php?target=pcr

The RT² Profiler™ PCR Array is a 96-well or 384-well plate for qRT-PCR analysis. Each array includes gene-specific Primer Assays for a thoroughly researched set of relevant, pathway- or disease-focused genes. It simultaneously profiles the expression of 84 pathway-specific genes, and five housekeeping genes. Each RT² Profiler™ PCR Array also includes a Genomic DNA Control (GDC) assay, triplicate Reverse Transcription Controls (RTC), and triplicate Positive PCR Controls (PPC).

For real-time detection, the RT² Profiler™ PCR Array is currently available for most QIAGEN, ABI, BioRad, Eppendorf, Stratagene, Cepheid, and Roche real-time instruments. Please refer to the link below, to determine which RT² Profiler™ PCR Array plate format is compatible with your instrument.

http://www.sabiosciences.com/manuals/PCRArrayGuide.pdf

These are pre-designed, validated qPCR assays optimized to measure genomic DNA sequence enrichment within chromatin immunoprecipitation (ChIP) samples. This technology expedites the identification of ChIP-enriched sequences within a 30 kb span (-20 to +10 kb) of every human, mouse, and rat transcription start site (TSS) annotated in the RefSeq database.

The ChIP-qPCR Assay System enables rapid, systematic whole genome interrogation of DNA-binding events for a DNA-binding protein of interest. This is due to SABiosciences' rigorously designed and validated qPCR primer collections, which amplify targets present in each consecutive 1kb genomic segment from -20 to +10 kb of every annotated human, mouse, and rat transcription start site (TSS). This approach generates a much richer, more quantitative data set, than the conventional gene-by-gene end-point PCR-based ChIP methods.

Each well of the PCR Array contains a single SYBR Green Real-Time PCR Assay. All of our Real-Time PCR Assays are wet-bench validated with our Master mixes to ensure target specificity, sensitivity, and reproducibility.

We like to start with cDNA from 10ng of total RNA per 25µL reaction.

Maybe. We design our gene assays based on the best Real-Time PCR characteristics of specificity, sensitivity and amplification efficiency. We wet bench validate every assay that we sell for these criteria.

Yes, stained sections can be used with the RT² PreAMP gene expression analysis system.

Yes.

The PreAMP system employs pathway-focused sets of 84 primer - each primer corresponding to a gene on each pathway-focused PCR Array. Each PreAMP primer mix supports 12 samples. To find the PreAMP Primer mix for your Pathway-Focused PCR Array, look here.

We provide validated primers for each gene in each well of the PCR Array. These same primers are also included in the PreAMP primer mixes. The primer sequences are not provided, as that is considered proprietary information.

While there is no way to check the quality of RNA from an FFPE samples, to determine the sample purity, calculate 260/280 & 260/230 ratios.

Protocols for FFPE block processing are also available in the user manual:

• Preparing Sections from FFPE Blocks: Cutting the blocks into sections
• Preparing Sections Mounted on Glass Slides
  

The oldest samples we had access to was 15 years. However, recommendation age limits are 20 years, though we caution the older the sample, the more likely RNA is degraded to a point where there is not enough recoverable effective template.

If you use our RT² FFPE PCR Array system with older samples and receive positive results, we would love to hear from you.

Our PreAMP technology aims to amplify non-degraded RNA to a point where enough effective cDNA template can be produced for PCR Array based analysis.

We recommend using the RNeasy FFPE kit, though QIAZol or other guanidinium isothiocyanate-based may work as well.

Yes, we offer RNA QC PCR Arrays for Human, Mouse, and Rat samples. http://www.sabiosciences.com/rnaqcarray.php

The optional RT² RNA QC PCR Arrays are designed to assess the quality of either human, mouse, or rat RNA samples before characterization with the RT² Profiler™ PCR Array as part of the complete PCR Array System. It contains a number of PCR controls that test for RNA integrity, inhibitors of reverse transcription and PCR amplification, genomic and general DNA contamination. Failure of any of these controls would otherwise confound SYBR Green based real-time PCR results by causing false negative or false positive results.

We are now offering the individual primer assays for genomic DNA contamination. Click here to visit the gDNA Primer Assay page.

Sizes requirements of FFPE blocks, sections, and number of slides are provided in our user manual.

No, you will need to order the FFPE RNeasy kit (Catalog Number: 73504) separately.

For both of these sample types, they generally are not fixed, so traditional sample PCR Array based analysis can be performed.

For LCM samples that are not fixed, we do offer the RT² Nano PreAMP technology for fresh and frozen samples yielding as little as 1 nanogram of RNA. You can find more information here.

In the slides, "RT No Pre-AMP" is meant to indicate PCR Array analysis on samples not undergoing the PreAMPlification step prior to analysis on an array. The "RT + PreAMP" indicates analysis on samples that have undergone pathway-focused PreAMPlifcation of cDNA prior to PCR Array analysis.

The RT step actually occurs on both samples - they only differ on whether PreAMP was performed or not.

Since we focus on gene expression analysis with our PCR Arrays, all of our protocols have been developed for RNA extraction, and so we do not have any recommendations for DNA extraction from FFPE samples.

Please visit our support page here.

By exploiting the properties of Proteinase K digestion at an elevated temperature, Xylene is not required in sample processing.

The files that you receive from your real time instrument should be excel-compatible files. From these files, you can extract the Ct values that can then be inputted into our free data analysis software (Excel or Web-based).

Our PreAMP technology has been designed (and this has been verified) to faithfully maintain the original gene expression profile during the PreAMP process. In a population of genes with both high and low expressers, preamplification amplifies all genes equally such that overall sensitivity of detection increases, while the differential patterning is maintained

The quality of the RNA isolated from FFPE samples will depend on the technique used to execute the archiving procedure and may depend on the age of the block and other less tangible variables. Standard fixation recommendations for the eventual purposes of gene expression analysis call for fixing tissue samples for 14 to 24 hours in 4 to 10% neutralized formalin as quickly as possible after surgical removal. Longer fixation times and longer periods of time from surgery to fixation lead to more severe RNA fragmentation, resulting in poor performance in downstream assays. It is also highly recommended to completely dehydrate the samples prior to embedding in paraffin. Previously archived samples may not have been processed in this fashion, and the details of how the process was actually performed may not be available.