qPCR Assay Database and new assays: The QGCL has a database of over 1,000 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.
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 electrochemiluminescence 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.
High resolution melt analysis is an excellent way to identify samples that fall into the same group. Examples where HRM analysis is applicable are identification of two or more members of closely related sequences (species of a common genus or closely related alleles of the same transcript), methylation, SNPs and more. If necessary, one member of each group can be sequenced to make specific sequence identification. HRM analysis is based on a saturating dye and the altered melt signature between 2 or more closely related sequences differing by as little as a single base. PCR amplicons are best if short, up to 250 bases but can be longer depending on the sequences being interrogated. The core director can help with questions. HRM is performed on our Roche LC480 real-time qPCR instrument and analyzed with provided software.
Samples- DNA samples should be provided at 25 ng/µl in nuclease-free H2O. It is critical that the samples be as clean of RNA or protein contaminants as possible as any contaminant could affect the melt characteristics of the sample DNA. Samples should be provided in the same tubes as described for RNA and DNA quantification.
Data analysis- Data will be provided in an Excel workbook. All melt curve figures and sample identifications will be provided.
We can screen human, mouse and rat samples for micro RNAs by real-time qPCR using Exiqon reagents and arrays. As with any screening protocol, it is critical that total RNA samples be prepared to include the smaller micro RNAs. Purification of miRNAs is not required but the proper kit should be used. Sample quality is always important but more so for this kind of screen. As with microarrays, an Agilent 2100 chip will be run with all samples to ensure RNA quality. Samples that fail this step will be returned to the investigator. Samples should be at 20-100 ngµl in nuclease-free H2O. Unlike expression analysis of individual transcripts, a single cDNA reaction is performed using ≈ 20 ng of total RNA. The entire cDNA reaction will be added to a single large PCR master mix volume and dispensed over 2 384-well plates bearing miRNA-specific primers (Exiqon) using liquid handling robotics. Six control small ncRNAs, a spike-in and inter-plate calibrators are included for each plate array.
miRNA hits identified by microarray or real-time qPCR has to be validated using a much larger sample set by real-time qPCR. Sample preparation is the same as for an miRNA screen above. The Agilent 2100 chip not only shows RNA quality but also the presence of miRNAs within the total RNA sample. As with the miRNA screen described above. A single cDNA reaction is run for each sample. The cDNA is then added to PCR master mix and dispensed in replicate wells over a 384-well plate followed by miRNA-specific primers. Samples should be provided at 25 ng/µl in nuclease-free H2O. Validation sample sizes are set at 16, 32 or 48 samples for 12, 6 and 4 primers per 384-well plate, respectively.
Data will be provided in an Excel workbook for each miRNA. Data will be analyzed by the director and supplied to the investigator in an Excel workbook. Analysis beyond that done by the core director will be the responsibility of the investigator.
Protein melt analysis is an inexpensive way to screen many small molecule compounds, aptamers, or other short molecules for their ability to bind to a protein. Protein melt measures an increase in fluorescence when a hydrophobic dye gains access to the inner core of a purified protein target as it denatures. The association of another moiety, if it interacts with a high enough affinity with the target protein, will affect the proteins melt curve through a slight change in protein structure that can be measured. The association of another molecule, if the small molecule interacts specifically with the target protein, that will affect the proteins melt curve through a slight change in protein structure that can be measured. By using a real-time qPCR instrument (Roche LC480), the melt curve can be recorded and analyzed utilizing software for this purpose. Contact the director of the QGCL for more information. Protein concentration would be in the 75-150 µg/ml range, depending on the molecular weight of the protein, and buffer conditions will depend on the conditions that are optimal for the target protein. In any case, the protein will have to be very pure, as with fluorescence polarization studies, to work.
Data analysis- Data will be provided in an Excel workbook. All melt curve figures and sample identification will be provided.
Protein quantification for complex samples is available using Meso Scale Discovery’s electrochemiluminescence reader.
Meso Scale- Assays provided by Meso Scale have 4-logs of linearity with detection limits in the low to sub-pico gram/ml range, depending on the assay. This assay can be multiplexed to as many as 10 assays/well on a 96-well Meso Scale plate. The ability to multiplex depends upon the expected antigen concentration range for each assay and potential cross-reactivity among Abs & Ags. The strengths are multiplexing of up to 42 samples in duplicate/plate, high sensitivity and a broad 4-log linear quantification range. The only drawbacks are the 6 hour total time to process a plate for the ELISA assays (mostly incubations and wash steps).
Data Analysis- Data will be provided in an Excel workbook with the appropriate figures and post-run analyzed data from the Meso Scale software. It is up to the investigator to complete data analysis in line with experiment requirements.
RNA and DNA Quantification
RNA- Total RNA (do not submit mRNA) should be as clean as possible. Perform an A260/280 and A260/230 measurement to ensure there is minimal protein (1.8 – 2.2) and salts contamination (1.7 or higher) that may interfere with PCR. Taq does not work well in the presence of foreign proteins, most of which will be those that bind to nucleic acids. RNA should be DNase I treated prior to submission. Not all assays cross intron junctions so DNA contamination can be an issue. Submit RNA in our 1.5 ml screw-cap tubes (for the robotics). We will provide tubes but if you need many you will have to order your own. Contact the core lab director for ordering information. For 384-well plates, the robot will add 2 µl of RNA sample/reaction or well. The recommended RNA concentration for submitted samples is 50 ng/µl (100 ng RNA/rxn). If RNA is limiting, dropping the concentration to 20 ng/µl will still work well. A final concentration of 10 ng/rxn can work with samples at 5 ng/µl but rare transcripts may fall below the lowest quantifiable limit of the assay (150 – 240 copies depending on assay PCR amplicon length). The lower limit of quantification is higher for RT-PCR than for PCR alone due to PCR inhibition by the reverse transcriptase (DNA binding). The most critical measure is the volume of sample submitted. The robots need more dead volume than when pipetting by hand. Therefore, we would like the first assay to have 20 µl with 15 µl volume for each succeeding assay. Samples with too little volume will not be run. Extra RNA can be returned to the investigator. Keep in mind that once an RNA solution has been normalized for loading using either a transcript assay or Ribogreen (Molecular Probes), additional transcript assays will not need to be normalized again if the original solution is used at a later date. Thus, submission of larger volumes can also be cost effective depending on the requirements of the project.
DNA- Genomic DNA should be clean of foreign proteins and RNA. Perform the same spectrophotometric analysis as above. DNA should have ratios of 1.8 – 2.0 and 1.7 or above, resp. As with RNA, the robot will add 2 µl of DNA/reaction. A concentration of 25 ng/µl is recommended although less can be used. Submit samples in the same tubes used for RNA.
Sample tube labeling and submission- The holders on the robot have a place for the tube caps. Please label the tube cap and side of the tube using a Sharpie pen. We run the samples blind primarily so label samples from 1 – N. If we are performing a ddCt analysis on samples, indicate which sample(s) to use as the calibrator sample for this analysis. The QGCL runs standard curves for unknown quantification unless we are running fact finding UPL or SYBR Green I assays. If you are in doubt how to proceed, contact the Core Lab director for clarification.
For both RNA and DNA samples, submit samples in a standard 1” freezer box labeled with the investigators name along with the sample submission form.
Both raw and normalized data will be provided within an Excel workbook for each investigator. The workbook will be expanded with each successive project and will thus be a repository for all data for that laboratory. The data from the qPCR instrument is analyzed by the core lab director following the run and an Excel macro is run to determine replicate homogeneity, DNA contamination levels and to array the final data in a user-friendly format. A graphic showing the standard curve and associated information is also proved for each transcript or gene assayed. A single normalized data tab will precede the raw data tabs and will contain the final data sets for each transcript or gene as well as for the transcript(s) or gene used for data normalization and the final normalized data set with standard deviations. Samples with values that fall below the lowest limit of quantification (LOQ), those with high DNA backgrounds or those with replicates outside assay QC limits will be marked by a color code that will be annotated. Data from mRNA and miRNA screens and validation experiments will be analyzed using GenEx software (Multid).