Quantitative Reverse-transcription PCR (Q-RT-PCR) in 384-well format


Overview

The UMGC offers Q-RT-PCR in 384-well format using the QuantStudio 5 Real-time PCR System real-time PCR instrument. Users may submit their own 384-well reaction plates to be amplified and detected on the QuantStudio 5, or they may elect to use the UMGC’s full-service Q-RT-PCR platform, including reverse-transcription to generate first-strand cDNA and Q-PCR based on Roche’s Universal Probe Library (UPL) or commercial Taqman probes.

Roche’s UPL technology is a library of 165 short hydrolysis probes designed such that virtually any gene will contain one or more appropriate probes. Designing a gene-specific UPL-based “Taqman” assay is thus quick and inexpensive, as only (inexpensive) primers are required that flank the hydrolysis probe.

The UMGC offers a service for design and functional verification of such Roche UPL Taqman assays, as well as a full-service, RNA-to-expression level analysis. This involves the iterative design, synthesis, and functional validation (PCR efficiency determination) of up to 3 primer- probe assays per target gene. Validation includes a multi-point determination of amplification efficiency.

Questions?


Contact Darrell Johnson: exprn@umn.edu.

Pricing

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Standard

Service Scale Unit UMN External
Design & Validation of Roche UPL primer-probe assay. 1-3 gene $81.27 $88.23
  4-8 gene $76.64 $93.06
  9-any gene $52.14 $61.10
Quantitative Real-time PCR Analysis with UPL Probe. 8-32 data poimt $9.45 $10.78
  16-32 data point $4.01 $4.64
  40-any data point $3.06 $3.57
Quantitative PCR. QuantStudio 5, Client-submitted Plate. 1-any plate of 384 $43.29 $50.19
Reverse-transcription of RNA. 1-any sample $14.62 $16.87
Taqman Assay Validation. 96-well Plate. 1-any plate of 96 $65.16 $75.11

 

Notes

Pricing valid for internal clients. PRICING FOR EXTERNAL CLIENTS WILL VARY FROM PUBLISHED RATE. Please contact UMGC staff to obtain a quote

Guidelines

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Submission

Contact exprn@umn.edu for project specifications.

Please review our Q-RT-PCR Project Submission Guidelines.

Complete the Assay Request Form, Project Request Form, or Project Continuation Form and email to exprn@umn.edu. Include a hard copy of the Assay Design Request Submission Form, Expression Run Submission Form, or QuantStudio 5 DIY Submission Form with plate.

Samples can be dropped-off at one of our laboratory locations.

1-202 Nils Hasselmo Hall (Minneapolis campus)

1-210 Cancer & Cardiovascular Research Building (Minneapolis campus)

20 Snyder Hall (St. Paul campus)

Deliverables

Client is emailed a data package. For assay design requests, this data package will contain efficiency information for passing assay designs. For expression run requests, this data package will contain the raw data as Cts, with mean and SDs of technical duplicate wells calculated. For 7900 run requests, this data package will contain the machine *.sds file, Ct data using machine optimized settings, and a copy of the amplification plot (and dissociation curves, if requested). Alternative settings are available upon request. Please contact staff for more details.

FAQ

How many technical replicates are run for each sample x assay combination?


We run two replicates (technical duplicates) for each “data point,” which is calculated as the mean of the duplicate reaction wells. Raw data returned include 1) the Ct of each duplicate, 2) the mean Ct of the duplicates, and 3) the standard deviation of the duplicates.

Is the ‘rate’ per data point the rate per replicate, or the rate per pair of technical replicates?


The rate per data point refers to the pair of technical replicates. You are not charged for each replicate independently, but for the mean value that is the “data point.”

Do you sequence amplicons during assay validation?


No, we do not routinely perform sequence validation, although this can be arranged and performed in the UMGC if so required.

How can I quantitate and QC my total RNA sample?


Simple UV spectrophotometry is adequate, and can be carried out on a standard UV spectrophotometer or a specialized instrument such as the NanoDrop. The UMGC also provides a quantitation service that you can use, if you do not have convenient access to a NanoDrop instrument. In addition to using the A260 to calculate RNA concentration, also record the 260/280 and 260/230 ratios in order to ensure that your samples are of high quality.

Do I need to perform QC on my sample using an Agilent Bioanalyzer?


We do not require electrophoretic QC of RNA. However, it is good practice, particularly when beginning a project using a new tissue source or extraction protocol, to validate that the quality of RNA is acceptable. Although the requirement for non-degraded RNA is not as strict for Q-RT-PCR as for microarray analysis, serious RNA degradation may interference with effective Q-PCR. The UMGC provides a QC service on the Agilent Bioanalyzer, if you do not have convenient access to such an instrument.

What is the turnaround time and timing of sample submission?


Q-RT-PCR assay design/validation turnaround options are 1-week or 3-weeks. Design/validation cycles begin each Wednesday at 10:00 AM, which is the deadline for submission of assay request forms. The deadline for the UMGC to return validation results to you is the end of the working day on Wednesday, 1 or 3 weeks later.

Q-RT-PCR expression assay turnaround is 1-week. Expression analysis cycles begin each Friday at 4 PM, which is the deadline for submission of project request forms and RNA. The deadline for the UMGC to return expression results to you is the end of the working day on Friday, 1 week later.