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Questions: (Click on topic of interest. Click on specific FAQ of interest to view the answer.)
SAMPLE PREPARATION: Maximizing the quality and yields of your RNA preps
REVERSE
TRANSCRIPTION: Choosing the right primers and controls
QUANTITATIVE PCR (qPCR): Designing and executing an effective qPCR experiment
DATA ANALYSIS: Converting data into meaningful results
SUPERARRAY qPCR AND qRT-PCR PRODUCTS: Trouble-free, innovative,
pathway-focused qPCR tools
Answers to SAMPLE PREPARATION FAQs
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How do I create a
workspace that is free of DNA
contamination, prior to carrying out a qPCR experiment? |
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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.
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How important is
the RNA purification process, for obtaining reliable qRT-PCR
results? |
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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.
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What is the key
technical challenge in isolating high quality RNA from cell or tissue
samples? |
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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 (10X µl of lysis buffer/mg of tissue or 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. Using larger volumes of lysis buffer to homogenize tissue samples
leads to better protection of the RNA from RNase degradation. Use a
combination of Trizol-based RNA extraction, followed by spin column-based
RNA purification. This typically yields an RNA preparation free of
inhibitory contaminants.
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What are the
most reliable methods for preparing high quality RNA from cell or tissue
samples, for use in gene expression analysis experiments? |
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We recommend the use of SuperArray’s RT2
qPCR-Grade RNA Isolation Kit (PA-001). 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 first homogenizing the tissue and performing
phenol/chloroform extraction with Trizol (Invitrogen), prior to utilizing the
SuperArray RT2 qPCR-Grade RNA Isolation Kit (PA-001).
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What are the key
technical challenges in isolating high quality RNA from Formalin-Fixed
Paraffin-Embedded (FFPE) tissue samples, and what are the most
reliable methods for preparing high quality RNA from FFPE tissue samples, for
use in gene expression analysis experiments? |
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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. We recommend the use of
SuperArray’s ArrayGrade FFPE RNA Isolation Kit (GA-023). The product uses a
modified RNA isolation procedure that reverses more cross-links than other
methods. As a result, the RNA is more available to the enzymes required
for gene expression analysis, resulting in superior gene expression
assay sensitivity.
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What is the
recommended solution in which to store RNA samples that are to be used as
templates for cDNA synthesis? |
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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.
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What testing
should be performed in order to assess the quality of an RNA sample? |
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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 4 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 SuperArray 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.
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What should I do
if I suspect that my RNA preparation contains RNase contamination?
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If you suspect that your RNA preparation contains RNase
contamination, re-purify the preparation over a spin-column based method,
such as SuperArray's qPCR-Grade RNA Isolation Kit (PA-001).
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How can I
avoid or remove genomic DNA contamination from the total RNA preparation?
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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.
When isolating RNA from tissue samples, it is recommended that the
tissue be subject to Trizol extraction (Invitrogen) prior to DNase treatment
and RNA isolation. In order to minimize DNA contamination in your RNA
preparations, and avoid the need for supplemental DNase treatments, we
recommend using the SuperArray RT2 First Strand Kit (C-03),
which includes a highly efficient genomic DNA elimination step before
reverse transcription.
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Answers to REVERSE TRANSCRIPTION FAQs
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What types of
primers are typically used for first strand synthesis in a reverse
transcription reaction? |
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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.
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What are the
differences between one-step and two-step reverse transcription-PCR
(RT-PCR)? |
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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.
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How can I
determine whether amplification occurs from mRNA-derived cDNA or from
genomic DNA contamination? |
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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.
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Answers to QUANTITATIVE PCR (qPCR) FAQs
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What is qPCR? |
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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.
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What are the common primer
and probe chemistries utilized for qPCR assays? |
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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.
SuperArray Bioscience 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.
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What qPCR/real-time thermal
cycler should I use for my qPCR experiments? |
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There are several manufacturers of high quality qPCR/real-time thermal
cycling instruments. These include 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.
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What is the purpose of the
ROX and Fluorescein dyes, also known as passive references, in qPCR
master mixes? |
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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.
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What negative controls are
typically included in qPCR and/or qRT-PCR experiments? |
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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.
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What positive controls are
typically included in qPCR and/or qRT-PCR experiments? |
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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.
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What guidelines exist for
choosing a housekeeping gene for normalizing qPCR results? |
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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 SuperArray 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.
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Why is 18S ribosomal RNA (rRNA)
used as a housekeeping gene to normalize sample-to-sample systematic
variation in the qPCR assays? |
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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.
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How does HotStart PCR help
minimize non-specific amplification events? |
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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.
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Why should I worry about,
and how do I minimize primer dimer formation, in my PCR assays? |
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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.
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What is a dissociation
curve, and why is it particularly important to run a dissociation curve,
following qPCR using SYBR Green chemistry? |
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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.
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How can I insure that
reaction volume is not lost due to evaporation during thermal cycling? |
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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.
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Answers to DATA ANALYSIS FAQs
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What is the delta Rn value? |
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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's manual.
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What is the threshold cycle
or Ct value? |
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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.
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Following thermal cycling,
reaction volume has evaporated from some of the wells of my qPCR assay
plate. What should I do? |
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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.
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Why are my qPCR Ct values
too low (< 12) in my qRT-PCR Assay? |
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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.
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Why are my qPCR Ct values
too high (> 35 or not detectable) in my qRT-PCR assay? |
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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.
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Why is my no template
control (NTC) real-time Ct value < 35 cycles in my qPCR Assay? |
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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.
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How can I determine whether
amplification occurs from mRNA-derived cDNA or from genomic DNA
contamination? |
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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.
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How can I predict the
percent qPCR signal due to contaminating DNA, for a given qPCR assay,
and its matching NRT control? |
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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% |
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Why do my qPCR
amplification curves or plots decrease in fluorescence intensity after
the saturation phase? |
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The optics of the qPCR instrumentation may be saturating due to improper
instrument settings. Please consult with your instrument manufacturer for
more details.
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Why do I see multiple
high-intensity peaks in my qPCR dissociation curve at temperatures less
than 70 ºC? |
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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.
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Why do I see multiple peaks
in the dissociation curve after my qPCR assay? |
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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.
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What is the standard curve
method for qPCR assay data analysis? How is the standard curve method
for qPCR assay data analysis performed? |
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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.
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What is the difference
between Absolute Quantification and Relative Quantification in qPCR,
using the standard curve approach? |
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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.
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What is the comparative or
ΔΔCt method for qPCR assay data analysis? How is the comparative or ΔΔCt
method for qPCR assay data analysis performed? |
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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).
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How do I determine the
amplification efficiency of my qPCR assay? |
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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
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Why do I see low, poor, or
sub-standard amplification efficiency in my qRT-PCR assay? |
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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.
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How do I determine the
linear dynamic range of my qPCR or qRT-PCR assay? |
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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.
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Answers to SUPERARRAY qPCR AND qRT-PCR PRODUCTS FAQs
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How do I download the User
Manuals for the SuperArray RT² qPCR and RT² qRT-PCR products? |
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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.
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Back to Top
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How do I obtain technical
support on the RT² qPCR and RT² qRT-PCR products? |
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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@superarray.net. Technical Support
representatives are generally available Monday to Friday from 9 AM to 6
PM EST.
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Why should I use a
SuperArray RT² qPCR SYBR Green master mix with the RT² qPCR
Primer Assays ? |
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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.
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What do I need to complete
a RT² qRT-PCR Assay? |
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You need: 1. A SuperArray 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 (Cat# C-03) for reverse
transcription.
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What RT² qPCR Primer
Assays are available for the RT² qPCR and RT² qRT-PCR Assays? |
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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.
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Can I obtain the sequence
of the RT² qPCR Primer Assay that I purchased? |
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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.
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Why are the RT² qPCR
Primers not designed to cross exon-intron junctions or boundaries? |
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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.
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How does SuperArray control
the quality of the RT² qPCR Primer Assays? |
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Each RT² PCR Primer
Assay is validated at SuperArray, 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.
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What does the RT² qPCR
Primer Assay Product Information mean when it says that it recognizes
another transcript of the same gene? |
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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
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Why do I need to identify
my real-time instrument model when placing my order for the RT²
qPCR Primer Assays? |
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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.
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Which qPCR instrument
should I use with your qPCR Primer Assays ? |
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Our RT² qPCR Primer
Assays may be used on any real-time instrument. SuperArray offers qPCR
solutions for the most popular qPCR instrumentation, including those
from ABI, BioRad, Stratagene. SuperArray
has written instrument-specific protocols for select instruments, which can be accessed at the
following link:
http://www.superarray.com/pcrarrayprotocolfiles.php
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What would happen if I used
home-made PCR master mixes or master mixes from other manufacturers with
the RT² qPCR Products? |
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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.
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What is the recommended
amount of input template for each RT² qPCR or RT² qRT-PCR
Assay? |
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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.
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Can the RT² qPCR
Primer Assays be used to validate results from SuperArray's GEArray
microarrays? |
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Yes, an RT² qPCR Primer
Assay corresponding to each gene on every one of SuperArray's microarray
products, or any other microarray product, is available.
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Why had my RT² qPCR
master mix been working well in the past, but now does not seem to be? |
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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.
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