|
|
|
Request password for FileShare
|
FAQs
1. WHAT THE DIFFERENCE OF TOTAL RNA REQUIRED FOR mRNA AND microRNA EXPRESSION PROFILING General speaking we require high quality of intact total RNA as start material for further process in MASR from researchers. For mRNA expression profiling, we recommend RNEasy Column purification, after RNA isolation by Trizol, to remove small RNA and contaminated protein. However, for microRNA expression profiling, we need whole total RNA without any column purification because of the nature of mature microRNA in size of 19~22nt and their precursors in size of 60~110 nt. BACK TO TOP 2 . WHAT TYPES OF OLIGO-MICROARRAYS ARE AVAILABLE IN THE MASR? The Microarray Share Resource of Comprehensive Cancer Center at Ohio-State University utilizes both Affymetrix and Custom Array Platforms. MASR provides the expression profiling and SNP genotyping on all Affymetrix commercially available products. In addition, the MASR also in-house built oligo microarray platforms available for mRNA and microRNA expression profiling. BACK TO TOP 3. WHAT WE DO AND WHAT IS YOUR RESPONSIBILITY? The research investigator provides high quality total RNA only. The facility further provides the in-house microarray fabrication, biotin-labeled antisense RNA (aRNA) target preparation, target/chip hybridization, chip post-hybridization signal detection, chip scanning, chip girding, data crunching, preliminary data analysis and data interpretation if needed. BACK TO TOP 4.WHAT IS THE PRINCIPLE OF EXPRESSION PROFILING TECHNOLOGY OFFERED IN MASR? The technology for expression profiling is a single color system on both Affymetrix and Custom Arrays. The oligo probes are in situ synthesis (Affymetrix 25mer) or spotted (Custom Array 65mer) and immobilized on the chip. The biological testing sample is further processed by linear amplification and biotin-labeling during in vitro transcription (IVT) to generate a single strand antisense RNA (aRNA) as a target for chip hybridization. The labeled aRNA on a single chip for hybridization and signal detection by Streptavidin-Alexa 647 in the post-hybridization process. The median normalized data from control and testing samples are compared to each other in fold change for further identification of differentially expressed genes. BACK TO TOP 5. HOW DO I DESIGN MY MICROARRAY EXPERIMENTS ? Since we have established that the chip to chip variance is low (4-8 %), it is most economical to prepare multiple biological samples and not request multiple chips for the same mRNA sample. Keep in mind that with 20K~45K spots, some spots will not hybridize equally on your control and treated chips because of technical reasons (minor defects, fiber etc.). For this reason it is essential that you repeat your experiment initially two times. You can then focus your initial analysis on the mRNAs that are altered in both your experiments. If you find that this initial experiment provides data that is promising, you most likely want to perform a third experiment to facilitate statistical studies. It is important to stress that it is not efficient to perform only one control and treated chip without a duplication because you are likely to spend significant time analyzing data that is artifactual. Typically, the facility has received samples comparing testing samples from control sample Of the thousands of samples have been processed we have noticed that labs have taken two strategies:
While we recognize that many of you have limited resources that motivate strategy 1, we strongly recommend option 2 despite its higher up front costs. If you have questions about your experimental design please send an email inquiry to Dr. Changgong Liu ( chang-gong.liu@osumc.edu ) -- BEFORE YOU PERFORM YOUR EXPERIMENT! BACK TO TOP 6. HOW DO I ISOLATE RNA FOR USE IN AN MRNA MICROARRAY EXPERIMENTS? The main objective is to generate RNA that is of high quality and sufficiently concentrated for use in a microarray experiment. Many researchers have found that RNA isolation with Trizol reagent gives good quality and quantity of RNA for these experiments. However, each type of starting material may generate RNA of different quality and no one technique is likely to work for all samples. Your data quality depends on your total RNA quality provided by you. BACK TO TOP 7. HOW DO I EVALUATE THE RNA QUALITY FOR EXPRESSION PROFILING ON MICROARRAY? RNA quality can be evaluated by visualizing the RNA on a gel or Agilent Bioanalyzer 2100, as well as by calculating the A 260 /A 280 ratio. On a denaturing gel or Bioanalyzer 2100 (or on an ordinary agarose gel in denaturing buffer) the RNA should appear as two bright distinct bands that represent the 28S and 18S ribosomal species. The 28S band should be brighter than the 18S band. Tailing of these major bands down the gel, or a background smear behind these bands that gets heavier at lower molecular weights may indicate degradation of the RNA. In these cases, it is best to isolate RNA from fresh tissue, as degraded RNA will produce high background and low signal intensities on a microarray. The presence of very sharp bands higher than the 28S ribosomal band can indicate the presence of excess DNA in the sample, which can be removed by treatment with RNase-free DNase I. The spectrophotometric ratio will also give an indication of the purity of the RNA, and should be as close to 2.0 as possible. Generally, ratios less than 1.7 indicate that the RNA may be contaminated with other material, and should be re-purified, perhaps run through a column. BACK TO TOP 8. WHAT SHOULD THE CONCENTRATION OF THE TOTAL RNA BE FOR SUBMITTING THE SAMPLE TO THE FACILITY? The concentration of the testing RNA should be 0.5 µg/µl minimal in DEPC-treated H2O. The facility requires 5~10 µg of total RNA for each testing sample(s). BACK TO TOP 9. WHAT SHOULD I DO BEFORE SUBMITTING THE SAMPLES TO THE FACILITY? Please make sure 1) the RNA quality has been properly evaluated and provide an agarose gel image with the RNA samples or request MASR for quality check on Agilent Bioanalyzer 2100, 2) complete the facility MASR service request form with the appropriate information. Failure to provide the complete information will delay the sample process on chips. BACK TO TOP 10. HOW SHOULD THE RNA BE SENT AND ADDRESSED? The RNA should be sent on dry ice via an overnight carrier for external users to the address listed on the facility homepage. All RNAs will be stored in -80 o C freezer at the facility. BACK TO TOP 11. WHAT ARE THE COSTS ASSOCIATED WITH A MICROARRAY EXPERIMENT? The costs of services are variable based on your affiliation with Comprehensive Cancer Center , for example, CCC member, non-CCC member on OSU campus and Off-campus users. Contact the facility directly for more detailed pricing information. BACK TO TOP 12. WHAT INFORMATION AND DATA DO I GET BACK AFTER AN EXPERIMENT IS DONE? You will get back all scanned chip images as .cel file, data file and .tif files. Also, you will be provided with the raw data in an Excel spreadsheet that has been crunched and exported from the image data and preliminary analyzed data with fold change of listed differential ly expressed genes. BACK TO TOP 13. HOW DO I RETRIEVE THE DATA? Data can be retrieved in several ways. Most commonly, an ftp site is set up on the computer of the CCC server with a private account for your data. You will be sent the IP address of the relevant computer, your ftp site is username and password. You can then access the site online to download the data to your own computer. BACK TO TOP 14. WHAT IS THE TURNAROUND TIME FOR EXPERIMENTS? |
