To assure RPPA assay generates the highest quality data requires samples to be prepared according to specific protocols and policies and to meet the specifications as outlined below.
Core assisted preparation of samples vs. investigator preparation of samples
Cell lines and other cell cultures. Core personnel or the investigator can prepare cell lysates. If investigators chose to prepare their own lysates it must be done with RPPA lysis buffer provided by the core using the specific protocol and instructions provided. ccurate protein concentrations must be determined for each lysate either by BCA or Bradford assays.
Tissues or tumor samples. Protein lysates from tissues or tumors requires preparation with a TissueLyser instrument in the core and with RPPA lysis buffer. Ideal tissue/tumor weight is a minimum of 15mg and weight needs to be provided by the investigator. There are two options: core personnel can prepare lysates or investigator can be trained and instructed to use the TissueLyzer in the c ore and prepare their own. Protocols and RPPA lysis buffer provided by core are required.
Body fluids (serum/blood or urine). Investigators will be required to collect blood or urine and process samples by specific protocols provided by the core.
Rare cells available only in small numbers. The core can perform RPPA with small numbers of cells as an isolated pellet. The low end range is dependent on the cell type, and this needs to be determined in consultation with the core director for each project. Investigators are required to collect and wash cells in PBS buffer, pellet cells by centrifugation, remove all PBS supernatant and flash freeze as pellets at -70C. An accurate cell count and total cell number in the pellet must be provided to the Core. Either Core personnel or Investigator can prepare cell lysates using Core RPPA provided lysis buffer and specific protocols. Protein concentration assays are not required.
Charges for RPPA Core services will be adjusted accordingly for core assisted sample preparations vs. unassisted.
Protein lysate specifications
Protein quantitation needs to be measured by recommended BCA or Bradford protocols and blanked with the RPPA lysis buffer used for protein extraction. Failure to use RPPA lysis buffer will lead to inaccurate protein concentrations that will affect RPPA results. For cell cultures and tissue/tumor extracts, the desired protein concentration before adding sample buffer is 1.1 - 3 µg/µl (mg/ml), and the optimal protein concentration of lysates for RPPA (final submitted protein concentration) is 0.5µg/µl (0.5mg/ml). For each experimental group, protein lysates should be adjusted to the same total protein concentration (0.5µg/µl). Please follow the protocols.
| Sample Types | Background | # of biological replicates |
|---|---|---|
| Cell lines or cell cultures | - | >=4 |
| Mouse tissues/body fluid | Pure background | >=5 |
| Mouse tissues/Body Fluid | Mixed background | >=8 |
| Human samples | - | >=10 (consult statistician) |
Submission of samples
An RPPA sample submission form must be filled out.
- For Core prepared lysates only sample identification is required plus information on cell numbers for cell pellets or weight for tissues/tumors.
- For investigator prepared lysates, the sample submission form also requires results of the protein concentration assays and standard curves and calculations.
Quality control of lysates by test spotting
A test printing of lysates with staining for total protein by Sypro Ruby is available to determine quality of samples for RPPA. This will estimate the accuracy of protein concentration and assess the quality of samples as viscosity can adversely affect spot quality and size.
Sample acceptance decision.
If samples do not meet required specifications, whether to proceed with the RPPA experiment will ultimately be at the discretion of the of Core director. Performance of the RPPA with samples that do not meet specifications will require an acknowledgement from the investigator of the risk of failure to obtain quality data and an understanding that Core charges will still need to be paid. A RPPA Sample Suboptimal Authorization Form needs to be signed by PI.
