| 1. How should I prepare my samples and how much starting material do I need? |
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Detailed information on how to set up your experiment is provided in the Protocol Section ("Reaction Set-Up"). Starting material can be either cDNA (two-step RT-PCR) or directly RNA (one-step RT-PCR). The amount of material that can be assayed per sample varies according to the cDNA synthesis protocol or the one-step RT-PCR mixture used by the customer. Essentially, the lower limit for starting amount of RNA depends on the sensitivity of the Reverse Transcriptase enzyme used. The UDSF staff has optimized some protocols to help customers who are using this technique for the first time. Please, again, refer to the cDNA Synthesis or to the One-Step RT-PCR procedures provided in the Protocol Section.
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| 2. How do I submit samples? |
| Once your plate is ready, please bring them directly to the DNA Sequencing facility, located in the BST L5 Rm 151. The UDSF staff will prepare your plate for loading on the real-time PCR machine and will assist you with the software interface in entering your samples and editing the cycler protocol according to your specifications. Please be on time for your reservation. After the run has completed, your raw data file will be uploaded in your lab's sequencing folder, so that you can download it on your computer and proceed to data analysis. The UDSF will provide the software for data analysis and help you with installation problems. |
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| 3. How do I reserve a real-time PCR run? |
| There are three sessions (9:30-12:30, 1-4, and 4:30-7:30) available for Opticon II or the ABI 7300. A Reservation for one of the three sessions is made through iLab. Once you log into iLab, click on the Schedule equipment tab then click view schedule for the instrument you would like to reserve. Highlight the time block you would like to reserve and complete the form. |
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| 4. How do I cancel my reservation and what is the cancellation policy? |
| To cancel a reservation, log into iLab and cancel the reservation. |
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| 5. Can I use plates and caps other than the specified BioRad or ABI ones? |
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No. The plates and caps from BioRad and ABI have been specifically designed to fit the heat block of their respective instruments. Using other types of plastic will compromise correct sample heating dynamics, heat uniformity and fluorescence detection. For this reason, samples submitted in plates other than the appropriate ones will not be accepted.
All of the required plates and caps are available for purchase from the DNA Sequencing Core facility.
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| 6. Do I need to include samples for a standard curve in each run? |
| We strongly recommend that you do so. There is a significant variability between runs, so that quantitation of the same samples will not yield the same exact absolute values in independent runs. Furthermore, a standard curve serves as internal control for the run conditions. |
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| 7. How many samples do I need for an accurate standard curve? |
| In theory, three samples are enough to identify a linear function. In practice, some samples can fail or result inaccurate because of pipetting errors. The UDSF recommends the use of at least 4 samples for the standard curve. |
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| 8. Do I need to include blanks in each run? |
| Definitely. The presence of blanks (samples that contain no cDNA or RNA) is as important as it is in any PCR experiment, in order to evaluate possible sample contamination. We recommend one blank per mastermix reaction (i.e. per primer pair). |
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| 9. What kind of DNA should I use as standard? |
| Any kind of DNA should work well. The DNA of choice should be very carefully quantitated by multiple spectrophotometric readings. DNA containing the gene of interest is an instinctive choice for standard DNA. This is a mistake. Standard DNA will be handled continuously and the risk of contaminating reagents, pipettors and PCR area with this DNA is very high. Our recommendation for standard DNA is to choose a plasmid DNA containing an insert (e.g. a short fragment of cDNA) from a gene that is of no interest for the lab research. Specific primers should be designed to amplify the standard DNA and used, in case of suspect results, to check for contamination. |
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| 10. What should I do if I am interested in recovering my samples after the run? |
| First of all, you want to make sure that your plate is labeled before you hand it in, so that it can be identified. You can pick it up any time after the run is finished up until one week later. All samples are stored in the UDSF fridge. |
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| 11. Which internal control gene should I use for normalization in my experiment? |
| This choice depends strictly on the experimental set up. Ideally, the investigator should examine the behavior of several housekeeping genes and make sure that their expression remains constant in the experimental system of interest. For more accurate results, more than one internal control gene can be used. |
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| 12. Can I quantitate more than one mRNA species in each well? |
| Yes. The Opticon II and ABI 7300 instrument are designed to work with technologies that require dual-dye detection systems, such as TaqMan and Molecular Beacons. |
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| 13. Where do I get the software for data analysis? |
| The UDSF customers can download the software for data analysis from this web site or borrow the CDs at the Facility. |
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| 14. Who can help me with data analysis? |
| The staff at the Core can assist with experimental design and data analysis. Please contact the core to setup a consultation. |
