Roche LightCycler 480
The UWBC Gene Expression Center offers qPCR services - utilizing the Roche LightCycler® 480 Real-Time PCR System - for microarray validation assays, gene expression studies and SNP analysis. Multiple levels of service are available to meet all campus researcher needs. Service options include:
- a full service where the user provides the RNA or DNA and the Center will design and optimize the assay(s) as needed
- user provides source material and primer/probes of interest and Center will perform assays as needed and
- user submits a ready-to-go plate that is run by GEC staff on the instrument.
Training on protocols / procedures are also available at $80 / hour.
Primer design and validation. Primer design is performed using Primer3 (http://frodo.wi.mit.edu/) and/or Roche’s UPL website. Once designed, primers are ordered from the UWBC DNA Synthesis facility and resuspended in TE to a 100 uM concentration. 10 uM aliquots of the primers are then used in conventional PCR on user supplied or commercially available genomic DNA target for primer validation. Primers that have demonstrated the ability to amplify gDNA in conventional PCR will then be examined for qPCR efficiency, using a dilution series of a positive control nucleic acid.
RNA QC and cDNA synthesis DNAse treatment of all RNA samples is strongly recommended. RNA samples are qc’d using the Agilent 2100 BioAnalyzer to verify sample integrity and the NanoDrop 2000 Spectrophotometer to verify sample concentration and purity. First strand cDNA is synthesized from intact RNA using SuperScript II (Invitrogen).
qPCR of RNA samples qPCR is carried out on RNA samples, using the Roche Light Cycler 480. Depending on experimental goals and conditions, either a SYBR Green or a Taqman probes based assay is used. Software included with the Roche LC 480 is used to quantify amount of target RNA present in samples, either as an absolute copy number or as quantification relative to an identified control.
If you are interested in qPCR service, or for more information, please contact GEC staff at firstname.lastname@example.org.
Roche Light Cycler 480 independent instrument usage
The Roche LightCycler 480 is available to use independently on a fee for use basis. Users must be trained by GEC staff prior to first usage. Cost of training is $80 / hour. Please contact GEC staff at email@example.com to schedule a training.
Note: Users must use Roche LightCycler 480 plates and sealers when running samples on the Roche LightCycler 480. These products are available from the Roche website.
Please use account# 55185396 when ordering from the Roche website to insure UW contract pricing.
Please direct any Roche related product or ordering questions to Beth Nigh at 1-630-319-0775, or email her at firstname.lastname@example.org