Nucleic Acids

qPCR Assay Database and new assays: The QGCL has a database of over 700 validated quantitative assays made and available for investigator use. The QGCL maintains all existing and new qPCR assay components. The investigator only pays for the assay once and succeeding investigators pay only the plate run charge for using an existing assay. The QGCL pays for future maintenance of all existing real-time qPCR assays. We can also make new qPCR assays as long there is good sequence information available. New real-time qPCR assays are designed by the director of the QGCL. Assay design begins with in silico investigation of the transcript/gene looking for splice variants, allelic isoforms, and highly related sequences. This information is used to guide the final design of the real-time qPCR assay. Four primers (2- forward & 2-reverse) are ordered for each assay around a fluorescent dual-labeled probe (hydrolysis-type). A long DNA oligo (PAGE purified) spanning the longest possible PCR amplicon is ordered to be used for assay quality control (QC) and for unknown quantification. Each new assay must have a lower limit of 10-20 copies by qPCR and a PCR efficiency of 93% or better. For microarray, miRNA or siRNA knockdown validation, we have the Roche UPL (Universal Probe Library). Primers are ordered for existing UPL LNA-based probes for the validation experiment. However, these assays only give a relative quantification to one of the samples (ddCt or dCt analysis) unlike the quantitative qPCR assays described above. Although the QGCL can run SYBR Green I based assays, and we have, we prefer probe-based assays for their increased template specificity. Further, validation of SYBR Green I assays is much more tedious (gel electrophoresis, sequencing and/or restriction digests). Template specificity validation will be the responsibility of the investigator. The QGCL can determine PCR efficiency and limit of quantification (LOQ) as per the other assay types listed above.

Proteins

The QGCL can set up and read any existing Meso Scale single or multiplex protein target ELISA utilizing our Biomek NXp robotic workstation with Thermo Cytomat labware hotel and Biotek ELx405 automated plate washer. For low sample numbers, investigators are encouraged to setup and run their own Meso Scale kit plates for which we can give advice. We can also assist in the development of novel assays. The most important assay components are a pair of antibodies that are specific for the target and recognize different epitopes on the protein. Meso Scale has single spot plates for this purpose that we can use to try multiple Abs in different combinations (for capture or detection) and read them on the MSD 2400 electrochemoluminescence reader. Charges will be determined by the number of hours it takes for assay development and supplies. We can also assist the investigator in performing assay development within their laboratory. Use of the MSD 2400 reader is available to outside users on per plate fee basis. Meso Scale will develop novel assays for a fee should that be desired.

For more information on quantitative protein detection systems, make an appointment to speak with the core director of the QGCL.