Real-Time PCR

Fluorescence-based real-time PCR is an established method for precise, efficient and rapid nucleic acid detection. It combines amplification and detection in a single step. The sensitivity of this method permits reliable detection of small amounts of starting template. Because the PCR product is detected using fluorescent dyes in real time, it is quantitative without  requiring the use of gel electrophoresis.   
 
Real-Time PCR Key Applications:
  • Gene expression analysis
  • microRNA and non-coding RNA analysis
  • SNP analysis
  • Genotyping/allelic discrimination

Instrumentation

Bio-Rad CFX96

The Research Core Facility has three Bio-Rad CFX96 Fast Real-Time PCR Systems. The CFX96 is a six-channel real-time PCR system that combines advanced optical technology with precise thermal control to deliver sensitive, reliable detection. The system’s solid-state optical technology (six filtered LEDs, each with a corresponding filtered photodiode) maximizes fluorescence detection for specific dyes in specific channels, providing sensitive detection for quantitation and target discrimination. Data is collected from all wells during data acquisition. At every position and with every scan, the optics shuttle is reproducibly centered above each well, so the light path is always optimal and there is no need to sacrifice data collection on one of the channels to normalize to a passive reference. Users can select multiple data acquisition modes, including a one-color fast scan for SYBR green. Thermal gradient features can be used to optimize reactions in a single run. The new CFX Manager software has advanced analysis tools for performing normalized gene expression. In addition, this system does not require fluorescein or ROX for instrument normalization.

Recommended Sample Volumes

CFX96 Fast

10-25 ul

 

Dye Calibration

Fluorophore

Channel

Plate Type

Cal Gold 540

2

Clear/White

Cy5

4

Clear/White

FAM

1

Clear/White

HEX

2

Clear/White

Quasar 670

4

Clear/White

ROX

3

Clear/White

SYBR

1

Clear/White

TET

2

Clear/White

Texas Red

3

Clear/White

VIC

2

Clear/White

 

Click here to download the CFX96 Fast Instrument Manual

Bio-Rad CFX384

The Research Core Facility has one Bio-Rad CFX384 Touch Real-Time PCR System.  The system's solid-state optical technology (5 filtered LEDs and 5 filtered photodiodes) provide precise quantification and multiplex target discrimination.  All other acquisition and analysis parameters are identical to the CFX96 Fast System.

Recommended Sample Volumes

CFX384 Touch

5-20 ul

 

Dye Calibration

Fluorophore

Channel

Plate Type

Cal Gold 540

2

White

Cal Orange 560

2

White

Cal Red 610

3

White

Cy5

4

White

FAM

1

White

HEX

2

White

Quasar 670

4

White

ROX

3

White

SYBR

1

White

Tex 615

3

White

Texas Red

3

White

VIC

2

White

Fees

Run Fees:

On Campus:  $25/run or $1200 annually
Off Campus:  $40/run

Instrument Training Fees:

On Campus:  $100/individual
Off Camus:    $150/individual

An annual account can be set up providing unlimited access to all four instruments for $1,200.00. This fee applies strictly to instrument time. Reagents and consumables must be purchased by the investigator. If you are interested in setting up an annual account, please contact Core staff. Billing is done at the beginning of each fiscal year and runs from July 1st through June 30th.

Core Facility technical staff will provide initial training (which includes using primer and probe design software, PCR reagent preparation, instrument setup and data analysis), as well as assistance with troubleshooting.

Access to primer and probe design software, as well as data analysis software is available at no cost to the investigator.

Scheduling

Reservations must be made in advance by signing up on the Brown Bear Calendar.

Instrument

Calendar

Password

Bio-Rad CFX96 Fast (1)

Brown Bear CFX96-1

realtime2018

Bio-Rad CFX96 Fast (2)

Brown Bear CFX96-2

realtime2018

Bio-Rad CFX96 Fast (3)

Brown Bear CFX96-3

realtime2018

Bio-Rad CFX384 Touch

Brown Bear CFX384

realtime2018


It is mandatory that you include your full name and a reachable phone number on the Brown Bear calendar when you schedule your appointment.

Rules for use

Basic Rules for Using the Real-Time PCR Systems in the RCF:

  1. All users must attend an instrument training session prior to their first use. These systems are end-user instruments, and users will perform all aspects of the setup. 
  2. Reservations on the Calendar are MANDATORY.  Reservations must be made in advance. Never put a plate in the instrument if you are not signed up to use it.  
  3. Include your full name and a reachable contact number on the Calendar and on the sign in sheet next to the instrument you are signed up to use.  It is not sufficient to put Lab "X".
  4. Remember to refresh your browser if the Calendar was left open.  If you do not refresh it, you may not see appointments that other users recently added, and this causes scheduling conflicts.
  5. Make sure that you use the instrument that you signed up for. If you are uncertain when you get to the Core, double-check on one of the PCs connected to the real-time PCR instruments. Again, remember to refresh the browser if the Yahoo Calendar was left open.
  6. Please be prompt and do your best to start your run on time. We understand that science is not exact and that delays do occur.  If you are late starting your run, you must notify everyone singed up immediately after you, or you must notify Core Facility staff.  Modify your appointment time on the calendar to reflect the delay.
  7. Reservations may be cancelled, but please be courteous to other users by canceling reservations as soon as possible and notifying Core Facility personnel. Failure to cancel an unused reservation will result in a minimum $25 charge even if you have an annual, unlimited account.
  8. Blocking off excessive amounts of time (i.e. all day or half of a day) and not fully using the time is not acceptable. You may reserve all three Bio-Rad instruments simultaneously for no longer than the time it takes to complete one run.  
  9. Users must sign the User’s Log next to the instrument when beginning a run. Please write legibly and include your first and last names, as well as your PI, Department and a reachable phone number.
  10. We recommend that you monitor the instrument for approximately 3 to 5 minutes after hitting the start run button (until you see the active status is "running"). Occasional errors may occur that can be corrected without any loss of data if caught early enough. If an error does occur, please notify Core Facility personnel immediately. If an error occurs after hours, please fill out one of the error incident report forms located to the left of the ABI 7900HT. It is often useful to use your phone to take a picture of any error messages present. 
  11. Please remove and discard your plates after the run. There is a Biohazard box located next to the FACS Calibur cytometer.
  12. If you are the last one signed up to use the instrument, please turn it off.
  13. DO NOT leave the instruments on over weekends or holidays, as this reduces the life span of our light sources.
  14. You are responsible for saving your data. We recommend saving all raw run files to a USB flash drive. There are shortcuts to the data folders on the desktops of each computer.  Do not save your data in folders other than those specified in training.

Notes:

  • If you fail to follow the rules above, it may result in the loss of data and Core privileges.  
  • Keep in mind that it is not appropriate for you to stop someone's run without contacting Core Facility staff first. Our contact information is located outside the door of BRI F6-13 (the real-time PCR room).
  • The Core will do everything possible to accommodate your needs; however, we ask that you be respectful of other users.

For more information, download a copy of the RCF Real-Time PCR User Guide and Policies.

Download the Training Guide.

Primer Design

Beacon Designer

Beacon Designer Software supports the design of SYBR Green primers, TaqMan Probes, Molecular Beacons, HRMA Primers, NASBA Assays, FRET Probes and Scorpions. All design is automated with optimal parameters set for specific assays. One of the advantages to this software is that it is possible to BLAST sequences and search for template structures from within the software.  The significance of this is that both of these searches are used during the design process so that regions exhibiting significant cross homology and template structures are avoided.  You can also design primers over junctions to avoid amplifying genomic contamination.

An online manual is not currently available; however, guides can be accessed through the software.  The software is located on two PCs in the Core Computer Lab, Room F6-27.

For a more thorough overview of the software, visit Premier Biosoft’s website.

Primer Design Considerations:

  • Keep in mind that primers designed for end-point PCR may not be optimal for real-time PCR.
  • Primer design is an important consideration for Real-Time PCR. Primers should be highly specific to your sequence of interest.  Blast your primers to check for specificity. 
  • Check primers for secondary structures, such as self-dimers and hairpins. As a general rule, the ΔG value for these structures should be more positive than -9.0 kcal/mole.
  • Order validated pre-developed primers from places like IDT, when possible.
  • If designing your own primers, use a software specific for qPCR and the chemistry (SYBR Green or TaqMan) you are using. Beacon Designer is available in the Core Computer Lab and the NCBI tool Primer BLAST can be accessed online.
    • It is good practice to design and order several pairs for each sequence of interest. Test them and choose the best one.
    • If you are having trouble finding the accession number that correlates to your gene, go to the IDT pre-developed assay page, query for your gene and retrieve the accession number from the returned results.
  • When possible, design your primers to span an exon-exon junction. This design reduces the risk of false positives from amplification of any contaminating genomic DNA, since the intron-containing genomic DNA sequence will not be amplified.
  • Consider the following when designing primers for SYBR Green Chemistry:
    • PCR product/amplicon size (80 to 150 is optimal).
    • The optimal melting temperature (Tm) of your primers is 60 to 64˚C. Ideally, the melting temperatures of the 2 primers should not differ by more than 2˚C so that both primers bind simultaneously and efficiently amplify the product.
    • The annealing temperature (Ta) is dependent upon the length and composition of your primers. In general, this temperature should not be more than 5˚C below the Tm of your primers.  If the Ta is too low, one or both primers may anneal to sequences other than your intended target, leading to nonspecific PCR amplification.  If the Ta is too high, you may see a reduction in reaction efficiency, as the likelihood of primer annealing is reduced.
    • GC content should be between 35-65% with an ideal content of 50%. Primer sequences should not contain regions of 4 or more consecutive G residues.
  • For more information on primer design or probe and primer design, visit IDT Designing PCR primers and probes webpage. 

MIQE Guidelines

MIQE (Minimal Information for Publication of Quantitative Real-Time PCR Experiments)

Guides

CONTACT US

Camille Abshire, MS, MDxT(AAB), CLS(LSBME)
Research Specialist, Genomics
cabsh2@lsuhsc.edu
318 675-4174

 

Rona Scott, PhD
Director, CMTV Genomics Core
Associate Professor, Department of Microbiology and Immunology
rscott1@lsuhsc.edu
318 675-6263