Frequently Asked Questions
1. What is the turnaround time?
Once we accumulate 1,000 samples from customers to accommodate a full set, the RPPA process takes 8 to 10 weeks. Service is provided on a "first-come, first-served" basis. We encourage you to submit samples as soon as possible.
2. How should I prepare my samples? Do you have special protocols?
We have protein extraction protocols posted on our Web site. Refer to the Protocols page. We can also provide lysis buffer and SDS sample buffer for your sample preparation. Please contact us to arrange a pick-up or shipment of the buffers.
*Please note that we have to receive and approve your paperwork before any buffers can be given out.*
3. After completion, what information and data can I expect to receive from the Core?
Your data set will be arranged in an Excel file. It includes raw data in log 2 value and normalized data in linear value. You will be able to generate bar graphs or other formatted graphs using normalized linear value. We also provide brief heatmaps with median centered log 2 value. These heatmaps represent visualization figures for your reference. Please consult your bioinformatics group for further detailed analysis.
4. How do you normalize the data set?
We normalize the data set by protein loading. Unlike western blot by b-Actin of GAPDH, we use the entire panel of antibodies to normalize protein loading.Briefly, the normalization is processed as follows:
- Convert raw data from log 2 value to linear value.
- Determine median for each antibody across the sample set.
- Divide each raw linear value by the median within each antibody to get the median centered ratio.
- Calculate the median from median centered ratio (from Step c) for each sample across the entire panel of antibodies. This median functions as a correction factor (CF.1) for protein loading adjustment. We consider the sample outlier if the correction factor is above 2.5 or below 0.25.
- Divide raw data in linear value (from Step a) by the correction factor (from Step d) to get the normalized linear value.
- We calculate the correction factor and provide you the normalized linear value. We also list the correction factor (CF.1) in the Excel file (the last column on the page of normalized linear value) for your reference to determine sample outliers.
5. How do we quantify protein expression and modification?
We use the approach of "Supercurve Fitting" developed by the Department of Bioinformatics and Computational Biology at MD Anderson Cancer Center to quantify protein expression and modification. Briefly, a "standard curve" is constructed from 5808 spots on each slide (one slide probed for one antibody). These spots include 5 serial dilutions of each sample plus 528 QC spots of standard lysates at different concentrations. Relative levels of protein expression and modification for each sample are determined by interpolation of each dilution curve to the "standard curve" (supercurve) of the slide (antibody).
6. If I have several RPPA data sets that were performed at different times within different RPPA assays, can I combine all my data together for further analysis?
As with any other biological assays, there are batch variations between each RPPA assay. At this time, it is not possible to combine the raw or protein loading normalized values. We are working with The Department of Computational Sciences and Bioinformatics to develop an approach for data merging. However, in any case, you should design constant internal controls that will be included within each experiment, to be used for data merging in future RPPA assays.
7. I have samples ready but they were prepared in the buffer for my western blot. Can I submit these samples for RPPA?
We encourage you to use the lysis buffer with our recipe. However, we accept samples prepared by the approach for western blot. Our experience demonstrates that samples prepared in RIPA buffer will work for RPPA. However, samples prepared from Urea buffer will make diffused spots that interfere with quantification.
8. What is the optimal protein concentration of the samples for RPPA? If my sample concentration is below 1 ug/ul, can you run this sample for RPPA?
The optimal protein concentration should be 1 ug/ul after SDS denature. We accept samples with a minimal concentration of 0.50 ug/ul (from cell lines), and 0.75 ug/ul (from tissue). If you have few samples with low protein concentration in your sample set, we suggest you not to adjust protein concentration against the lowest one. Otherwise, you will lose the majority of the signals.
9. How much volume of each sample do you need for RPPA?
We require to have at least 40 ul of each sample to accommodate the entire panel of 150 antibodies.
10. Do I need to prepare replicate samples for RPPA?
There are two kinds of replicate: biological replicate and technical replicate. Please design replicates according to your study. We do charge replicates as separate samples. We perform multiple technical replicates using our standard lysates across the slides on different positions. These replicates will function as quality control for each antibody in RPPA. We release the result from those antibodies which pass our quality control.
11. Do I need to select antibodies for RPPA?
No, we have a Standard Panel of Antibodies that we use for each set. Please refer to the Antibody List and Protocols page. You do not need to select antibodies from the Standard Panel, we will perform the entire panel of antibodies on this list and release the result from 150 antibodies that passed our quality control. Please note that we charge 60% overhead for submissions outside of MDACC. Please refer to our Services and Pricing page.
12. If the antibody against the protein of my interest does not exist in your list, can I provide you with an antibody?
Yes, you can provide your own antibodies. Since we do not have experience on your antibodies, it may take several runs to get the optimized dilution for RPPA. You will need to provide 30ul of the antibody along with the data sheet and Western Blot results.We charge $750 per sample set for each supplied antibody. Please note that each antibody in our list is validated for RPPA application. The antibodies you submit have not been validated, thus, we can not guarantee the result.
13. If I would like you to validate my antibody, do you provide such a service?
Yes, and we charge $750 per antibody. To request antibody validation, please submit a set of samples together with your western blot result with your antibodies and densitometry measurements on each band. We will perform RPPA on your samples. Please provide your antibodies together with the source information, such as host-rabbit or mouse, company and catalog number, etc. The validation will be judged on the following criteria:a) Single band on western blot with right molecular sizeb) Acceptable dynamic range on the specific protein manipulated by chemical or genomic approachesc) Good correlation between western blot and RPPA (r > 0.7)d) Acceptable Signal/Noise ratio on RPPA
14. I have a set of tissue samples to be submitted for RPPA analysis. How much tissue do you need from each sample? Do you have protocols to extract proteins from tissue?
Our experience demonstrates that 10 – 15 mg of frozen tissue (approximately the size of a grain of rice) for each sample is required to process RPPA for the entire panel of antibodies. We prefer to have your tissue samples prepared in our lysis buffer. Please refer to the protocols on the Antibody List and Protocols page.
15. Do you provide service to extract protein from tissue for RPPA?
Yes. Refer to our Services and Pricing page.
16. I have a set of clinical samples. Can I submit them for RPPA analysis?
Yes. You MUST delete patient sensitive information, such as Medical Record Number, Name, etc., before you submit. We will not accept samples with patient sensitive information.
17. I am in the process of submitting a grant application and/or manuscript which will involve RPPA analysis. Do you have any information I can refer to in my grant application?
Yes. Please refer to the Education and References page.
18. I am a research investigator outside of MD Anderson Cancer Center. How should I pay for the RPPA service? Do I need to get a Purchase Order Number from my institution?
Yes, we will need you to complete all information on the submission form and include a valid purchase order number. Our financial office at MD Anderson Cancer Center can make exceptions on a case by case basis if a PO# cannot be attained in a timely manner; full payment is due no later than 30 days after receipt of invoice. You can also pay by credit card too (please inquire for our credit card authorization form).
19. How should I send my samples to you? What is your physical address? What is your shipping address?
Please refer to our Getting Started page.
20. Do I need to validate my result from RPPA?
Yes. RPPA provides you a result of cell signaling networks in a kind of "screen" mode. You need to validate and confirm your result by other technical approaches.