Real-Time PCR

Background

The recent introduction of fluorescence-based techniques to PCR has allowed the development of real-time PCR, also called qPCR or kinetic PCR, which is widely used in gene expression analysis either as a primary method or as a confirmation of other methods such as microarray analysis. Real-time PCR is also the most commonly used method to monitor the efficiency of gene silencing by RNA interference. Other applications include measurement of DNA copy number and transgene copy number, determination of viral titers, and single nucleotide polymorphism genotyping.

Unlike conventional PCR that uses an end-point analysis of the amplicon, real-time PCR is based on the detection and quantitation of the PCR product as it is made, using either fluorogenic specific probes (such as Taqman probes) or double-stranded DNA binding fluorescent cfxdyes such as SYBR Green. Early in the PCR amplification process (exponential phase), conditions are optimal and the amount of double-stranded DNA theoretically doubles at every cycle. Therefore, during this exponential phase of the reaction, the fluorescent signal increase is directly proportional to the amount of PCR product
( Figure 1)                                                        


Instrumentation

The MCBF is equipped with a Bio-Rad CFX96 Real-time PCR Detection system capable of using rapid cycling protocols and fast chemistries to produce results in about an hour. The solid-state optical technology (six filtered LEDs & six filtered photodiodes) can discriminate up to 5 targets in a single reaction well, and has one channel dedicated to Fluorescence Resonance Energy Transfer (FRET) detection. The CFX manager software has built-in data analysis modules with automatic baseline subtraction and threshold setting.

In addition, a Bio-Rad iCycler iQ5 is available (96-well plate format) which combines a thermocycler unit  and an optical module that reads fluorescence. This machine is very versatile and has PCR multiplexing capability since the optical module can collect data for up to 4 different fluorophores within the same well.

Fees and Requirements:

                    
 To Use the iQ5 or CFX96 instruments:

  • You must take the introductory course in qPCR offered by the MBCF
                                          
  • To schedule training: Contact us at 423-439-8096 or via email at mbcf@etsu.edu


 Equipment Use Fee

  • CFX96 or iCycler: $25 / run ETSU; $50 / run non ETSU
                            
  • Eppendorf 5070 robot: $20 / full plate
       

 Primer Design by the MBCF

  •  Design primers-probes
                      
    • $75 - includes 1 free re-design
    • $100 non ETSU
    • multiplex designs: 2=$200 3=$300
                                
  • You must provide the Genbank Accession number of your gene of interest 
                               
  • We strongly recommend that you sequence the amplicon you obtain to make sure this is the expected product
       

 Reaction Setup

  • Unless the plate is sealed prior to bringing to MBCF, you must use a dUTPase system (see us for details if needed)
                                   
  • The MBCF provides the SYBR Green, if needed
                              
  • You must bring your own supplies (tips, tubes, gloves)
        

 Disposables

The MBCF sells disposables for use in qPCR, such as:

  •  Individual plates $4.85 
                   
  • Individual optically clear tapes $1.36 
                               
  • Pipet tips for the Eppendorf 5070 (50 μl/box of 96) $14.00
     
  • qPCR Supermix (Invitrogen, Bio-Rad, Qiagen, Promega)
        

Standard Curve

Real-time PCR data are analyzed after setting an arbitrary threshold that must intersect the signal curve in its exponential phase. The point at which the curve crosses the threshold is called Threshold Cycle (CT). The higher the starting copy number of the nucleic acid target is, the sooner a significant increase in fluorescence is detected, and the lower the CT value.

Quantitative analysis of unknown samples commonly uses a calibration/standard curve representing CT values as a function of Log amount/copy number of starting material  

In "perfect" reactions, the number of amplicon copies doubles at each cycle, and the characteristics of the resulting standard curve are:

- PCR Efficiency = 100%
- Correlation Coefficient = 1.000
- Slope = -3.300

For accurate quantitation of unknown samples, the standard curve should be as close to a "perfect" curve as possible, like in the example presented in ( Figure 2), and all test samples should be in the range of the standard curve.

Melt Curve
 
Applicable to assays using a fluorescent dye (SYBR Green) as detection method. A given piece of double-stranded DNA melts at a precise temperature which is primarily dependent on its base contents and length. As the temperature increases, the PCR product denatures and releases the associated SYBR Green, causing a sharp decline in fluorescence. The melt curve is obtained by plotting the negative first derivative values of the fluorescence intensity as a function of the temperature, which results in a peak.

The melt curve is a good indication of how clean the PCR product amplified is; ideally a single, narrow peak ( Figure 3 ).

The real-time PCR technology is widely and increasingly used, because it offers significant adv antages such as high sensitivity, wide quantification range, good reproducibility, and high throughput capabilities; it has become a critical tool for basic research and molecular diagnostics.

Guidelines

The MBCF offers on a regular basis a 2 hour introductory class to qPCR. It is mandatory to take this class once if you wish to operate our machine. In addition to this tutorial, our staff can help you with different aspects of your qPCR project such as primer design, practical help, troubleshooting, etc... The MBCF is also available to run samples for you. In this case, you do not need to take the course. For more information, please contact us at MBCF@mail.etsu.edu

Here are some guidelines to get you started:



Sample RNA

In real-time RT-PCR experiments, using high quality total RNA is crucial.

  •   Good ratio OD 260nm/280 nm (1.8)

  •   No degradation (28S/18S rRNA ratio of 2:1 on an agarose gel)

  •   No/low contamination with genomic DNA (DNase treat your RNA)

Suggested Total RNA isolation kits:

- Qiagen RNeasy- Mini Kit includes an on-column DNase digestion using RNase-free DNase set

http://www1.qiagen.com/default.aspx  

 - Ambion - RNAqueous Mini and Midi Kits   http://www.invitrogen.com/site/us/en/home.html  
 - For high throughput: Promega Automated SV96 Total RNA Isolation System can be used on the Beckman Biomek 2000

robot, present in the MBCF. This kit is designed to isolate total RNA from cultured cells but we have used successfully isolated

RNA from animal tissue (rat brain)

For small size samples or low abundance transcripts, an RNA linear amplification step can be performed prior qRT-PCR analysis. Several companies propose kits in which the entire transcript population present in a sample is amplified between 1,000 and 5,000 times. This amplification is linear so the differential gene expression is preserved. For more information, visit the following websites:


Reverse Transcription

Most of the time, first-strand cDNA synthesis is done using oligo(dT) and/or random hexamers, but gene specific primers can also be used

Note:
We keep in stock IDT Ready-Made Oligo(dT) and Random Hexamers
Many commercial RT kits are available, the following kits are available in MBCF Stock:

  • Invitrogen Superscript II
  • Invitrogen Superscript III
  • Promega GoScript *in HELIX FREEZER*

  Primer design - The most important determinant for accurate/specific results

Predesigned primer sets for many genes are available. Qiagen www.qiagen.com/GeneGlobe and Invitrogen www.invitrogen.com   (TaqMan primer/probes) are just two companies that offer validated primers.

We can design qPCR primers for you but here are some recommendations if you prefer doing the design yourself.

  • The primers should span a DNA region with no secondary structures. DNA folding programs are available online to use for free.  Suggested link: http://www.bioinfo.rpi.edu/applications/mfold/     Make sure to include folding/annealing temperature (55°C-68°C) and salt concentrations of the assay (commonly 50 mM Na+; 3 mM Mg2+)

  • Ideal amplicon size: 50 to 200-250 bp

  • If working on cDNA: primers should span at least one intron. Suggested link: http://www.ensembl.org/

  • Database including information regarding exon/intron junctions, transcript variants, protein information, etc...

  • Ensure that the designed primer pair is specific for the gene of interest. We strongly recommend doing a BLAST search ( http://www.ncbi.nlm.nih.gov/blast/) on these primers

    Primer design softwares

  • Free webtools at http://www.idtdna.com; Primer Quest, mFold, OligoAnalyzer 3.0

  • GeneRunner

  • Vector NTI license site on ETSU Campus

  • Visual OMP4 (DNA Software Inc.) purchased by the MBCF; for primer pair and probe design; integrated DNA folding module; single-plexing, and multiplexing. Contact us for details.

Primers

  • No formation of primer dimers

  • No runs of 4 or more same nucleotides, especially G

  • No more than 2 G or C in the final 5 nucleotides (primer 3’ end)  

  • Match Tms as close as possible (0.5 C if possible)

  • GC content of 40-60% is recommended

Endogenous Control Genes for Normalization

To minimize variations in the amount of input material (DNA, cDNA) used in the qPCR reaction, the results for your target gene should be normalized to those obtained for an endogenous control gene. The "perfect" control does NOT exist but should be validated for your system/model. Ideally, this control gene should be constitutively expressed in all cells, preferably expressed at similar levels as the gene(s) of interest and, most importantly, its expression should NOT change in your application.

Examples of commonly used controls: GAPDH, GUSB, β2-Microglobulin, TATA Binding Protein (TBP), Transferrin Receptor (TfR)

   The MBCF has, available to users, primer sets for human GAPDH and b2-Microglobulin, and for rat GAPDH, assays for which the qPCR conditions have already been optimized (SYBR Green assays).

Standard Curve

The standard curve can be dilution series of:

  •  DNA/cDNA- Arbitrary/ Relative number

  •  Cloned PCR product- Copy number = Absolute number

  •  If amplicon size < 100 bp, a purified synthetic single-stranded oligonucleotide of the same sequence as the PCR product can be used- Copy number = Absolute number

  • T7-transcribed RNA template - accurate quantitation?

Important: For an accurate quantitation of unknowns samples, make sure all test points are in the range of the standard curve

Quantitation Strategies

Different quantitation strategies are currently used:

    Absolute Quantitation

        - Standards must be accurately quantitated

        - Requires multiplexing; amplification of the internal control and of the gene(s) of interest is performed in a single tube

        - Final result is usually reported relative to a defined unit (copies per ng of total RNA, per genome, per cell or mg of tissue         - Used for example for viral load determination and inter-lab comparisons

   

  • Relative Quantitation

      - Results usually reported as a ratio Gene of Interest/Endogenous Reference

       -  Best used for gene expression studies 

   

  • Comparative Quantitation

      - Requires a thorough preliminary assay optimization

       - Samples all prepared identically

       - No standard curve; Calibrator sample used as 1X standard in every run

       - Suitable for high throughput applications

       - Mathematical determination of fold increase / decrease in gene of interest expression across samples using :       

                * No normalization

                * Normalization using at least one reference gene and with efficiency correction (REST method) or without real-time

                   efficiency correction (DDCt method)

   

For more information about quantitation methods, visit http://www.gene-quantification.info/

qPCR Setup

Manual: takes about 2 hours to setup a full plate

Automated: using the Eppendorf 5070 ($20/plate), plus tips

Side-by-side comparisons of manual vs. automated plate setups have shown very comparable results in term of C T values and reproducibility between replicates ( Figure 4, Figure 5 )

How do I set-up my SYBR Green assay? click here

Note: Prepare a sufficient volume of PCR mixture for the number of wells needed, including 4+ extra wells if the Eppendorf 5070 is used to setup the plate; minimal extra volume required, regardless of the number of wells used

Suggested Run

Run samples in triplicate

3 blank wells (No template)

3 negative control wells (RT minus)  at least once when using a new set of primers

4-5 point standard curve (each in triplicate)

1-27 samples in triplicate

If you need to run multiple plates for the same primer set, make sure to put at least one common sample on all plates for accurate comparison

  

After the first run using a new set of primers, we recommend to check the amplicon:       

                  - size on an agarose gel

                  - sequence

iQ/Cfx software  - After running your assay on the either of the real-time PCR machines, data analysis must be done in your laboratory. Software is available at no charge for download on your machine


 
To Use iCycler

  •  Must take the introductory course to qPCR offered by the MBCF at least once. You can re-take the course as a refresher as needed.

  •  Must schedule time with MBCF

  •  Must have an MBCF account

  •  Do NOT analyze your data on the MBCF machine, software (Windows) is free to all users - Collect Data on Zip disk or  burn to CD-R

How can the MBCF aid you with your qPCR project?

We can help you with different aspects of your qPCR project depending on your needs. We can:

    - design your primers for a small fee

    - help you get started with the design of your assay

    - sell you disposables for qPCR

    - provide endogenous control primers we have designed & tested

    - show you how to use the iCycler

    - help you setup the plates using the Eppendorf 5070 robot - for a small fee

    - troubleshoot your qPCR assays and help you optimize your experimental conditions

    - and, if you are running short on time and technical expertise, we can set up and run your assays for you. Ask us for details!