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 willl 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).