Guide to Utilization of Microarray System
Guide to Utilization of Microarray System
1. System Utilization Flowchart 1-1. Send an e-mail to the Microarray Center with a hybridization request (application for utilization) Contact the Microarray Center by sending an e-mail stating your request for a microarray experiment. The address is skikuchi@nias.affrc.go.jp. The e-mail must contain the five following items. Assignment Number (if you have it) Requested time of hybridization Number of samples Your e-mail address Abstract describing purpose of your research There is a limit to the number of RNA samples you can submit with your application. Up to twelve RNA samples may be submitted when the one-dye method is used, or six pairs if the two-dye method is used. Please consider allowing for repeated experiments in a single "application" (example: experiments conducted twice for each of six different types of samples when using the one-dye method). We ask that e-mails with applications for utilization of the facilities be sent at least one month prior to the desired testing date. 1-2. When a reply is mailed from the Microarray Center When you receive an e-mail in reply acknowledging "receipt of application for experiment" and accepting the application (including Assignment Number if you had not had it before you sent the e-mail stating your request to the Microarray Center), please send the following four items (a-d) to the address provided. a. Quality-checked RNA (checking procedures described below) b. Document with attached photograph of the electrophoresis-checked total RNA and description of the RNA in the photograph (single A4-sized page) c. Document with photograph of results of reverse transcriptase (RT)-PCR (real-time PCR or other quantitative PCR) (single A4-sized page; format of this document corresponds to that of b.) d. CDs for return of data (three 640-MB discs) At the time all of the items a-d are sent, enter the required information for the RMOS "Microarray System Utilization Form" page, and submit the form on line. A password for the "Microarray System Utilization Form" page will be furnished in the e-mail reply. Send RNA using an express parcel delivery service especially for frozen materials, such as the "Cool Takkyuubin" service, specifying morning delivery (Saturdays and Sundays excluded). Send the four items (a-d) to the address given below. Address for Submission: Dr. Shoshi KIKUCHI (Microarray Center) Head of Gene Expression Research Team, Department of Molecular Genetics, National Institute of Agrobiological Resources Kannondai 2-1-2 Tsukuba, Ibaraki 305-8602 Japan <Use an express parcel service specifically for delivery of frozen materials, and specify morning delivery.> 1-3. Notification from the Microarray Center with the scheduled time for analysis After the Center receives the four items (a-d, including RNA samples and the data information) as specified in the 1-2, the Center set up a hybridization schedule of the RNA samples. Each applicant is informed of the date that the analysis will be performed, and then the microarray analysis is carried out. 1-4. Data from Microarray Center is returned on CDs After the hybridization experiments are carried out, the results are scanned with an FLA-8000 scanner (Fujifilm) and quantified with ArrayGauge (Fujifilm), and then is copied to the CDs and returned to the applicant(s). |
2. Guide to Sending RNA Samples 2-1. Tubes to be used (Eppendorf tubes) Please submit samples in 1.5-ml Eppendorf tubes (non-screw-cap: the type suitable for array experiments). 2-2. Instructions for labeling the Eppendorf tubes Using an oil-based permanent marker, write the "Tube Label" (see note below) both on the top of the cap and on the side of the tube. If also attaching an adhesive label, be sure to use a label that cannot be removed easily. Moreover, write the "Tube Label" information on the Eppendorf tube itself before attaching the label. Be sure to carry out the step of writing on the tube itself, as this information becomes crucial should the label happen to become separated from the tube. (Note: The "Tube Label" should contain the sample number and a description identifying the sample named in the "Tube Label" heading in the "Microarray System Utilization Form" described in 1-2) 2-3. Precautions When Sending Samples Send samples precipitated in ethanol. Before mailing, securely wrap the capped portion of the Eppendorf tubes with plastic film (e.g. Parafilm) or other suitable wrapping to prevent leakage of the sample (ethanol leakage). Attention: If upon arrival at the Microarray Center a tube is found to be leaking or to have leaked, either the remaining contents in the tube will be analyzed as is, or the sample will not be used at all. |
3. RNA Quality Control Guide Please be sure to perform all of the three following quality checks on all RNA samples for which microarray analysis is desired. Submitting results of all three methods for checking the RNA quality are one of the essential conditions for acceptance of the application. Enter the OD value data of the RNA for which analysis is desired in the " Microarray System Utilization Form ," send the application on line, and mail the documents ("total RNA QC" and "RT-PCR QC") with the photographs of the electrophoresis gels to the Microarray Center. 3-1. Measurement of OD values (to be entered on the "Microarray System Utilization Form") Enter numerical values in the "OD260/280," "RNA Density," "t=," and "m=" columns. "t=" is the column used for indicating total RNA, and "m=" is the column used for indicating mRNA.For example, if using total RNA with the one-dye method, no entry need be made in the "m-" column. However, if using mRNA with the one-dye method, or if employing the two-dye method (when only mRNA is being handled), enter the necessary information in both the "t=" and m=" columns. 3-2. Electrophoretic check of total RNA (Separate Form 1; a single A4-sized sheet) With all RNA for which array analysis is desired, use 10 ƒÊg total RNA in agarose gel containing 1.1% formaldehyde, perform electrophoresis, and photograph the results. After downloading and printing out the form "Total RNA QC" (a separate Form 1; a single A4-sized sheet), attach the photograph to the page and mail it in. In this case, describe which RNA samples correspond to the RNA in each lane in the photograph (i.e., which lane contains each RNA sample entered on the "Microarray System Utilization Form") so as to permit us to match each RNA sample shown on the gel with its corresponding "Tube Label." 3-3. RT-PCR check (Separate Form 2; a single A4-sized sheet) Carry out reverse transcriptase PCR using a gene that is commonly expressed in all RNA samples, both the RNAs as control and the RNA samples with which you want to compare the control RNAs, confirm with electrophoresis, photograph the gel. Then download and print out the form "RT-PCR QC" (Separate Form 2; a single A4-sized sheet), attach the photograph, and mail it in. Before mailing, describe how the RT-PCR check was carried out using the gene (e.g. the gene you used; the DNA sequence of the gene-specific primers you used, the type of PCR [normal RT-PCR or real-time PCR] you used; the PCR condition you used, etc.). To discover genes suitable for the RT-PCR quality control check, select a number of candidates from among the genes thought to exhibit constant expression (such as housekeeping genes) and conduct actual confirmation with RT-PCR. This procedure allows the selection of optimal genes for the purpose. However, genes that can be used for the purposes of 3-3 in certain experiments are not necessarily suitable for the same purposes in the other experiments, even when for the same purposes of 3-3. |
4. Labelling Selection Guide 4-1. Selection of the one-dye or two-dye method For a single experiment, please select either (not both) the one-dye method or the two-dye method (experiments using the two different methods are not conducted simultaneously). Refer here for help in selecting the one-dye or two-dye method. <Related Data> Differences in Fluorescence Levels for Cy3 and Cy5 4-2. Number of samples One-dye method: Up to 12 RNA samples can be submitted for one experiment Two-dye method: Up to 6 pairs (12 RNA samples) can be submitted for one experiment. Please consider allowing for repeated experiments in a single "application" (example: experiments conducted twice for each of six different types of samples when using the one-dye method). 4-3. Labeling instructions Whether selecting the one-dye or two-dye method, use the indirect labeling method. The AtlasTM Glass Fluorescent Labeling Kit (Clontech K1037-1; http://www.clontech.co.jp) is used for indirect labeling. For more detailed information on labeling methods (both indirect labelling and direct labelling), see here. For information on the data obtained by the Microarray Center using direct labelling and indirect labelling, see here. <Related Data> Differences Between Direct Labeling and Indirect Labeling Methods Differences Between Total RNA and Poly A+RNA Microarray System Utilization Form Once you understand the content of this guide to utilization of the system, go to the "Microarray System Utilization Form," and enter the necessary information. At the same time that you fill out the Utilization Form, send the four following items (1-4) by an express parcel service to the address given below. Address for Submission: Dr. Shoshi KIKUCHI (Microarray Center) Head of Gene Expression Research Team, Department of Molecular Genetics, National Institute of Agrobiological Resources Kannondai 2-1-2 Tsukuba, Ibaraki 305-8602 Japan 1. Sample RNA 2. CDs for return of data (three 640-MB discs) 3. Documentation with attached photograph(s) of the gel electrophoresis for the total RNA for all included RNA samples 4. Documentation with photograph(s) of gel electrophoresis after RT-PCR Enter Microarray System Utilization Form |
