RPPA CORE | BCM | CPRIT 2016
Reverse Phase Protein Array (RPPA) Policies and guidelines for sample
preparation
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.
1) 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. Accurate 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 Core 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.
2) 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
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RPPA CORE | BCM | CPRIT 2016
should be adjusted to the same total protein concentration (0.5µg/µl). Please follow the
protocols.
3) Recommended sample replicates.
Sample size (number of samples per experimental group) for RPPA project depends on the
project goal, sample types, and previous data/knowledge. The exact number of samples for the
project should be discussed among the investigator, core director, and statistician during the
project initiation phase. As a starting point, we recommend the following biological replicates
for each sample. The Core routinely spots all protein lysates in triplicates of arrayed slides.
These are technical replicates.
Sample Types
Background
Cell lines or cell cultures
# of biological replicates
>=4
Mouse tissues/body fluid
Pure background
>=5
Mouse tissues/Body Fluid
Mixed background
>=8
Human samples
>=10 (consult statistician)
4) 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.
5) 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.
6) Sample acceptance decision.
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RPPA CORE | BCM | CPRIT 2016
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.
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(RPPA) Policies and guidelines for sample preparation