Quantifying RNA With QRT-PCR: 2-3 Hours

Quantifying RNA with qRT-PCR

Is my gene of interest present? Does the level of my gene of interest change due to an experimental condition? To what degree does the treatment change the level of expression of my gene of interest? Previously, these questions were addressed strictly by the technique of Northern blotting. Microarrays can also address these questions on a larger scale, but qRT-PCR can be used to focus on an individual transcript. Microarray results are typically conrmed by qRT-PCR and vice versa. qRT-PCR has many advantages over Northern blotting. The rst obvious advantage is time. A typical Northern blot experiment can take anywhere from 3 days to 2 weeks, depending on the abundance of the target transcript. qRT-PCR can go from RNA to answer in just a few hours. qRT-PCR requires only nanogram quantities of RNA, whereas Northern blots require microgram quantities. Northern bolts use mostly radioactive probes, while qRT-PCR uses uorescent reporters. qRT-PCR, however, does require a special thermal cycler with uorescent detection capabilities. Northern blots can be performed with much less equipment. Only Northern blots can tell you the size of the full transcript and the integrity of the RNA sample. Real-time, quantitative RT-PCR (qRT-PCR) is the current method of choice to answer the following questions: Northern blots require methods like densitometry or uorimaging to relate the relative abundance of an individual transcript. qRT-PCR data are related in terms of numbers, allowing easy analysis of fold change in an experiment. Both methods rely on control genes to normalize data from sample to sample. The control gene should not change with the treatment. The ratio of your gene of interest to the control gene will normalize differences in sample loading from well to well. qRT-PCR can be used to quantitate a target transcript in a given sample through the use of a standard curve generated from standard samples in the same experiment.
qRT-PC R offer s a to the questions quick answer : Is my transcrip t presen Does t? the leve l of my under a tran n experim ental co script change To wh ndition? at d level ch egree does m y transc ange? ript
Perform qRT-PCR Treat Cells with Effector Isolate RNA
Analyze Data
23 hours
RNA to Electrophoresis
Transfer RNA to Membrane
Probe Blot
Expose Blot
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Analyze Data
314 days
Comparison of Northern blotting procedure to qRT-PCR procedure.
www.promega.com techserv@promega.com
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Detection Methods for quantitive, real-time amplification
DNA Binding Dyes
The earliest method developed for real-time PCR involves adding ethidium bromide to a PCR then monitoring the change in uorescence due to the accumulation of double-stranded PCR product using a ber optic cable and a spectrouorometer (1). The most popular dye for this mode of qPCR is SYBR Green. The SYBR Green dye is added during the PCR, and binds to the minor groove of double-stranded DNA. The signal increases as the amount of double-stranded DNA increases (2). within the hairpin anneals to the newly synthesized PCR product, disrupting the hairpin and separating the uor and quencher. A similar approach involves a specic PCR primer linked to a double-stranded oligonucleotide probe. The probe consists of a uorescently labeled strand and a quencher-labeled strand and has sequence that is complementary to the PCR product. In the absence of PCR product, the two strands anneal, the uor and quencher are in proximity, and uorescence is quenched. As the PCR product accumulates, one strand anneals to the PCR product instead of the second strand of the probe, freeing the uor from the energyabsorbing effects of the quencher. These probes involve intramolecular hybridization, which is kinetically more favorable than intermolecular hybridization.
Hydrolysis Probes
Hydrolysis probes are labeled with a uor at the 5-end and a quencher at the 3-end, and when the two reporters are in proximity, the uorescent signal is quenched. During the annealing step, the probe hybridizes to a synthesized PCR product. The resulting probe:target hybrid becomes a substrate for the 53 exonuclease activity of Taq DNA polymerase, which degrades the annealed probe (3) and liberates the uor. The uor is released from the effects of the quencher, thereby increasing uorescence.
Simple Hybridization Probes

The use of simple hybridization probes involves either two labeled probes or one labeled probe and a labeled PCR primer. In the rst approach, the energy emitted by the uor on one probe is absorbed by a uor on the second probe, which hybridizes nearby. In the second approach, the emitted energy is absorbed by a second uor that has been incorporated into the PCR product as part of the PCR primer. Both of these approaches result in increased uorescence of the energy acceptor and decreased uorescence of the energy donor. The use of hybridization probes can be simplied even further so that only one labeled probe is required. In this approach, quenching of the uor by deoxyguanosine is used to bring about a change in uorescence (5,6). The labeled probe anneals so that the uor is in close proximity to guanine residues within the target sequence. As the frequency of probe annealing increases, the level of uorescence in the reaction decreases due to deoxyguanosine quenching. The advantage of simple hybridization probes is their ability to be multiplexed more easily than hydrolysis and hairpin probes through the use of differently colored uors and probes with different melting temperatures (reviewed in 7).
Hairpin Probes
Hairpin probes contain inverted repeats separated by a sequence complementary to the target DNA. The repeats anneal to form a hairpin structure, with a uor at the 5-end and a quencher at the 3-end. With the reporters in such proximity, the uorescent signal is quenched. The hairpin probe is designed to preferentially bind to the target DNA rather than retain its hairpin structure. As the reaction progresses, the probe anneals to the accumulating PCR product and, as a result, the uor and quencher separate. The uor is no longer quenched, and the level of uorescence increases. These probes are difcult to design properly; secondary structure needs to be mapped out carefully to ensure that the probe anneals preferentially to the PCR product. Recently, hairpin probes have become part of the PCR primer (reviewed in 4). In this approach, the stem-loop structure of the hairpin probe is attached via a nonampliable linker to the specic PCR primer. Once the primer is extended, the complementary sequence
References
1. Higuchi, R. et al. (1992) Biotechnology (N.Y.) 10, 4137. 2. Morrison, T. B., Weis, J.J. and Wittwer, C.T. (1998) BioTechniques 24, 954-962. 3. Holland, P.M. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 727680. 4. Bustin, S.A. (2002) J. Mol. Endocrinol. 29, 2339. 5. Crockett, A.O. and Wittwer, C.T. (2001) Anal. Biochem. 290, 8997. 6. Kurata, S. et al. (2001) Nucl. Acids Res. 29, e34. 7. Wittwer, C.T. et al. (2001) Methods 25, 43042.
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Promega RNA Analysis Notebook

Plexor Technology for Quantitative, Real-Time Amplification
H N H O H N iso-dC H N Ribose N H O N N N iso-dG N Ribose
The Plexor Systems work by measuring a reduction in a uorescent signal during amplication. Amplication occurs through the use of two primers, one containing a uorescent tag and the other containing a modied base. As amplication proceeds, uorescence is reduced by site-specic incorporation of a uorescent quencher opposite a complementary modied base in one of the primers. When the quencher is in proximity to the uorescent tag the uorescent signal is reduced (quenched). After PCR, a melt analysis can be run to provide an internal control for the nal assay design or to expedite troubleshooting during development. Multiplexing is also possible with the Plexor technology. Multiplex assay design is simplied by the use of a web-accessible Plexor Primer Design Program specically engineered for multiplex assay design.
iso-C an d base pair iso-G cannot with th e nat bases A, C, G or ural T

N G N Ribose
O H N H N H N C N Ribose O N H
Novel Base-Pairing drives Plexor

The Plexor technology offers a new method for qPCR. It takes advantage of the specic interaction between two modied nucleotides to achieve quantitative PCR analysis. The two novel bases, isoguanine (iso-dG) and 5-methylisocytosine (iso-dC), form a unique base pair in a double-stranded DNA molecule.
Iso-bases are incorporated as easily as standard dNTPs by DNA Polymerases Johnson, S.C. et al. (2004) A third base pair for the polymerase chain reaction: Inserting isoC and isoG. Nucleic Acids Res. 32, 193741.
An excellent resource for quantitative PCR is: Bustin, S.A. (2004) A-Z of Quantitative PCR. International University Line Biotechnology Series, La Jolla, CA
Courtesy of International University Line (orders www.iul-press.us)
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Plexor Chemistry into Quantitative PCR.
Fluorescent Reporter iso-dC Taq
Primer Annealing and Extension

Taq
iso-dGTP Dabcyl
Incorporation of Dabcyl-iso-dGTP
Quenching of the uorescent signal by dabcyl during product accumulation.
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Fluorescence Quenching
To perform uorescent quantitative PCR using the new Plexor technology, one primer is synthesized with an iso-dC residue as the 5-terminal nucleotide and a uorescent label at the 5-end; the second primer is unlabeled. During PCR, the labeled primer anneals to the template and is extended, becoming part of the template during the next round of amplication. During subsequent rounds of amplication, the complementary iso-dG, provided in the Plexor Master Mix as dabcyliso-dGTP, pairs specically with iso-dC. When the dabcyl-iso-dGTP is incorporated, the proximity of dabcyl and the uorescent label effectively quenches the uorescent signal as the product accumulates.
A.
First reported use of iso-bases in a real-time qPCR application: Sherrill, C.B. et al. (2004) Nucleic acid analysis using an expanded genetic alphabet to quench uorescence. J. Am. Chem. Soc. 126, 45506.
Exponential phase Amplification threshold
Plexor Reactions produce C t Values

By directly coupling uorescence detection and thermal cycling, real-time PCR measures the change of uorescent signal (in relative uorescent units, RFU) at every cycle. The initial uorescence level of the labeled primers is high in Plexor System reactions. As amplication product accumulates, the signal decreases. Amplication data present a characteristic three-phase curve (Figure to right, panel A). Data obtained during the exponential phase gives the most consistent estimate of the amount of starting material. Thus, the amplication threshold is set within the exponential phase at a uorescence level where all amplication curves exhibit the most signicant signal decrease. The point at which the amplication curve crosses the threshold is the cycle threshold (Ct) of the sample. The Ct values from a dilution series of known DNA quantity are used to generate a standard curve, which is used to quantify samples with unknown amounts of DNA (Panel B).
B.
andard s for st to Ct d converte ve and cur standard ration of concent wns unkno ned determi
Plexor Reactions produce Cts and Standard Curves. Panel A. A representative amplication curve, that shows the relative uorescence units (RFU) at each cycle of the reaction. The cycle threshold (Ct) is set at 15% of the maximum uorescence decrease and is indicated by a horizontal line across the graph. Panel B. A standard curve generated from amplication curve data shown in panel A. Data were generated with a Bio-Rad iCycler Instrument and analyzed with the Plexor Analysis Software.
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Quenching of the uorescent label by dabcyl is a reversible process. Fluorescence is quenched when the product is double-stranded due to the proximity of dabcyl and the uorescent label. Denaturing the product separates the label and dabcyl, resulting in increased uorescent signal. Consequently, thermal melt proles can be generated by allowing all product to form double-stranded DNA at a lower temperature (approximately 60C) and slowly ramping the temperature to denaturing levels (approximately 95C). The gure to the right illustrates a melt curve from a Plexor Reaction with the empirically derived melting temperature (Tm). Product length and sequence affect the Tm. Consequently, the melt curve characterizes amplicon homogeneity in the selected wells. Nonspecic amplication can be identied by broad peaks in the melt curve or peaks with different Tm values.
Specificity Confirmed with Melt Curves
Thermal melt curve of data. The melting temperature was empirically determined by plotting the change in uorescence with temperature (dRFU/dT) versus temperature and calculating the temperature at which the biggest change in uorescence occurs. Data were generated with a Bio-Rad iCycler Instrument and analyzed with the Plexor Analysis Software.
Whats a Ct?
PCR is only truly quantitative when measurements are taken during the exponential phase, when reagents are not limiting and the change in uorescence is proportional to the accumulation of PCR product. The concept of the threshold cycle (Ct) is at the heart of accurate and reproducible quantication using uorescence-based RT-PCR (1). Fluorescence values are recorded during every cycle and represent the amount of product amplied to that point in the reaction. The more template present at the beginning of the reaction, the fewer the number of cycles it takes to reach a point at which the uorescent signal is rst recorded as statistically signicant above background (2). This point is dened as the Ct, and will always occur during the exponential phase of amplication. This value can be translated into a quantiative result by constructing a standard curve (3). References
1. Higuchi, R. et al. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplication reactions. Biotechnology (NY) 11, 102630. 2. Gibson, U.E., Heid, C.A. and Williams, P.M. (1996) A novel method for real time quantitative RT-PCR. Genome Res. 6, 9951001. 3. Bustin, S.A. (2000) Absolute quantication of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 16993.
What is amplication efciency?

Under ideal conditions, the amount of PCR product doubles each cycle. If there is a doubling at each cycle, the reaction is said to be working at 100% efciency. qPCR amplications should operate as close to 100% efciency as possible. Efciency is related to the slope of a standard curve plotted as Ct vs. log concentration. The equation for determining efciency is:
Efficiency = 10
n formatio more in For tion amplifica egarding y, see: r c efficien of
) AZ .A. (2004 nal Bustin, S PCR. Internatio titative ology Quan techn y Line Bio Universit olla, California J a Series, L
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-1 x 100%
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lt One me = One product
Impure have e products w x o falling tra melt pro uld ducts on the de either side sired p o roduct. f
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The Plexor reaction incorporates dabcyl-iso-dGTP opposite any iso-dC residue encountered. Therefore, quenching can occur with any primer synthesized with an iso-dC base. This naturally leads to multiplex qPCR. To demonstrate the multiplexing capability of Plexor Systems, reactions were set up in monoplex, duplex and triplex formats using two targets, broblast growth factor receptor 1 (FGFR1) and matrix metalloproteinase 1 (MMP1) and a control gene glyceraldehyde-3phosphate dehydrogenase (GAPDH). A concern during multiplexing is how to account for vastly different copy numbers of targets and control genes. The Plexor Technology reduces the concentration of control gene primers at least 2-fold to lessen the effect of amplication of the control gene on targets. In the example shown, Ct values for GAPDH typically occur about 8-9 cycles earlier than Ct values for either target at any total RNA concentration chosen. A change of 1 Ct represents two-fold more template when the reaction is at 100% efciency. In this case, the GAPDH is at least 2829 (or 250500-fold) more abundant than the targets FGFR1 and MMP1, but as can be seen in the curves, has no major affect on the quantitation of FGFR1 or MMP1, either separately or together. All reactions produced very good r2 values and amplication efciencies. Multiplexing the assey had only a minor effect on the Ct values compared to the monoplex Ct values.
Plexor reactions can do more than one color
A.
39 36 33 30 27 Ct 24 21 18 15 12
FGFR1
(FAM Reporter)
Plexor S ys you to r tems allow and con un targets trols same w in the ell!
FGFR1 FGFR1 + GAPDH FGFR1 + MMP1 + GAPDH
log pg Total RNA Template
B.
39 36 33 30 27 Ct 24 21 18 15 12
MMP1
(CalFlour Red 610 Reporter)
MMP1 MMP1 + GAPDH MMP1 + FGFR1 + GAPDH
C.
40 35 30
GAPDH
(JOE Reporter)
Ct 25
GAPDH
20
GAPDH + MMP1
15
GAPDH + MMP1 + FGFR1
10
Accurate quantitation of three targets in monoplex, duplex and triplex format using the Plexor One-Step qRT-PCR System. The indicated quantity human total RNA was assayed for the targets broblast growth factor receptor 1 (FGFR1), matrix metalloproteinase 1 (MMP1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reactions were set up with one Plexor Primer set, two Plexor Primer sets, or three Plexor Primer sets as indicated in each panel. Cts were obtained from all three conditions, and average Ct plotted against log picograms of template. Data are presented for FGFR1 Plexor Primers labeled with FAM (Panel A), r2 values ranged from 0.999 to 1.000 with amplication efciencies from 99 to 102%. Data from MMP1 Plexor Primers labeled with CalFluor Red 610 (Panel B), r2 values ranged from 0.995 to 0.999 with amplication efciencies from 101 to 102%. Data for GAPDH Plexor Primers labeled with JOE (Panel C), r2 values ranged from 0.999 to 1.000 and amplication efciencies ranged from 99 to 102%. FGFR1 and MMP1 primers were used at 200nM nal concentration. GAPDH primers were used at 100nM nal concentration. Data were generated with an Applied Biosystems 7500 Real-Time Instrument and analyzed with the Plexor Analysis Software. Plexor Primers were obtained from Biosearch Technologies.
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Bringing Plexor Technology to Your Lab
Use the Plexor Web Design Program for multiplex experiments. Order Plexor Primers from licensed oligo suppliers.
Design Your Assay
Choose the appropriate Plexor System and order.
Download the Plexor Technical Manual specific for your instrument.
Run Your Assay

Assemble reactions as directed in one Reagent Manual shipped with the Plexor System. Program your instrument using instructions in the Technical Manual.
Download Plexor Analysis Software and install on your PC.
Analyze Your Assay
Analyze data with Plexor Analysis Software as directed in your instrument Technical Manual.
Export raw data from real-time instrument.
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Import raw data into the Plexor Analysis Software.
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Choosing the Right Plexor System
Total RNA
Use your reagents for making cDNA
Plexor Two-Step qRT-PCR System Protocol available at: www.promega.com/plexorresources Cat.# A4051 10 Reverse Transcription reactions and 200 qPCR reactions
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Analyze RNA Directly?
Make cDNA, Analyze
Protocol System qPCR Plexor t: a sources available /plexorre om omega.c www.pr 4011 Cat.# A eactions qPCR r 200
Plexor One-S tep qRT-PCR System Protocol available at: www.promega.co m/plexorresourc es Cat.# A4021 200 qPCR reac tions
Each Plexor System is shipped with the appropriate reagent manual
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Programming your Instrument
rument the inst Plexor Program tting up se prior to ns for the first ns reactio ent instructio e trum time. Ins saved for futur can be reactions. Plexor
Instrument specific technical manual
Download Plexor instrument manual
ructions in ollow inst instrument F ic your specif regarding al manu g your programmin nt. instrume
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Technic instruct al Manuals prov ions for: ide Progra mm Export ing instrument ing raw da Import ing data ta Plexor s oftware into Data analysis Trouble s For all s hooting upported insturum real-tim e www.p ents go to:
romega.c om/plexo rsources
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Setting up Your Assays
Nucleic acid sample
Plexor Primers
Plexor System appropriate for your nucleic acid sample.
Set-up Plexor reactions as specified in the supplied Plexor reagent manual.
All qRT-PCR-based Plexor Reactions use 2-step cycling: 95C denaturation 60C hybridization and extension 40 cycles
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Run on preprogrammed real-time instrument.

Data Analysis
he Follow t your ions in instruct specific trument rding ins ega manual r lysis. data ana
Instrument specific manual gives instruction on how to use the analysis software
View amplification curves or melt curves for single wells, columns, rows or entire plate by simple selection.
Get C ts and Tms
Export data to Excel spreadsheets
Set melt thresholds.
Create screen shots of data with a simple command.
Identify unknowns for quantitation on standard curve.
Identify standards for standard curve.

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qPCR You use your own reagents for cDNA synthesis
Plexor qPCR System 200 reactions A4011
Two-Step qRT-PCR Generate cDNA, then perform qPCR

Plexor Two-Step qRT-PCR System 10 Reverse Transcription reactions 200 qPCR reactions
One-Step qRT-PCR Go from RNA directly to qPCR in one tube

Plexor One-Step qRT-PCR System 200 reactions A4021
A4051
Get Fast, Accurate Answers from Scientists

The Promega Worldwide Technical Service Group, Field Applications Specialists, and Distributors are committed to providing you with the highest quality service available to help you be successful. Each of these individuals has an extensive background in molecular biology research, hands-on experience with Promega products, and training in problem solving and troubleshooting. Additionally, the full resources of our R&D, Quality Assurance and Production Scientists are available to help increase your laboratorys productivity. Contact Promega Technical Services directly or through your Branch Ofce at: techserv@promega.com Online product support and information including references, protocols and questions and answers are available in our product reference section at: www.promega.com/applications/prtn_exp Also visit our Technical Resources web site at: www.promega.com/techserv for a wealth of support information on Promega product applications.
assistance Technical for is available p in the every ste cess! Plexor pro
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Quantifying RNA With QRT-PCR: 2-3 Hours

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Quantifying RNA with qRT-PCR