Introduction Reverse transcription and real-time PCR using FastLane Cell Kits are performed with a fast and convenient multi-step procedure that can be easily automated for applications such as high-throughput gene expression analysis. While automated workflows enable efficient processing of multiple samples simultaneously, there are frequently steps within the protocols that may introduce unintended standing times for assay components. Additionally, time constraints may require the operator to temporarily pause the procedure and store the samples until a later period.
This study describes an optimization of the procedure used for FastLane Cell Kits, including tolerated stopping points and optimal component storage conditions.
Results and discussion
Stability of cell lysate
In the standard FastLane Cell Kit procedure, cells are lysed, RNA is stabilized, and gDNA is eliminated during an incubation step for 5 minutes at room temperature, followed by a 5 minute incubation step at 75°C. The stability of the cell lysates under extended storage conditions was determined by introducing break points in 2 independent runs; one prior to the 75°C incubation step and another following the heat treatment. Lysates were stored for up to 24 hours at 4°C or –20°C. Following the break point, the protocol was continued and the lysates were stored at –80°C. Four targets genes (ACTB, NFKB, PPIA, and INPP5D) were examined in 2 cell lines (HeLa and HepG2). Analysis by RT-PCR was performed to determine the effect of the break points on the observed CT values (see figure
Determination of HeLa and HepG2 cell lysate stability).
Break points introduced prior to the 75°C incubation step resulted in only minor shifts in the observed C
T value when lysates were stored for 1 hour at 4°C or up to 24 hours at –20°C. However, strong C
T shifts were measured in samples with prolonged storage at 4°C. Notably, the target INPP5D was not detected after storage for 2 hours at 4°C, which suggests the effect is target specific.
Storage of the lysates after the 75°C incubation step had no discernable effect on the observed C
T values. Even after the samples were incubated for 24 hours at 20°C, no change in C
T values were observed.
Stability of real-time PCR reaction mix
Multiplex, real-time PCR reaction mixtures consisting of lysates prepared using the FastLane Cell Multiplex Kit, QuantiTect Multiplex RT-PCR Master Mix, and the primer and probes were assembled and allowed to stand for a pre-determined time period (40 minutes, 3 hours, 6 hours, or 24 hours) across a range of temperatures (4°C, 20°C, or 30°C). In all experiments, an initial incubation of 40 minutes was included to simulate the time required for a typical automated PCR reaction setup. Three gene targets (ACTB, OAS1, and NFKB) were examined in HeLa and HepG2 cell lines.
All targets were detected using the QuantiTect Multiplex RT-PCR Kit. No C
T shift was observed when reaction mixtures were incubated for 40 minutes at 20°C in the FastLane lysates (see figure
The effect of temperature on reaction mix stability). However, incubation periods of greater than 3 hours at room temperature or above resulted in C
T shifts in some targets and cell lines. For example, the target NFKB could not be detected after 6 hours of incubation at room temperature in both cell lines. Similarly, the target OAS1 was not detected after 3 hours of incubation at 20°C in both cell lines. Detection of this target likewise failed in HeLa cells when reaction mixtures were incubated for 24 hours at 4°C.
For all targets, incubation at 30°C for 2 hours resulted in significant shifts in C
T values (see figure
The effect of 30°C incubation on reaction mixture stability). The magnitude of the effect was depended on the target identity and cell line.
Optimizing the concentration of TaqMan Gene Expression Assays
Multiplex analysis using the FastLane Cell Multiplex Kit, which includes QuantiTect Multiplex RT-PCR Master Mix, delivers sensitive and reliable quantification without optimization. However, in some cases (approximately 10%) the use of TaqMan Gene Expression Assays (ABI) with the FastLane Cell Multiplex Kit can result in a small linear range. As the QuantiTect Multiplex RT-PCR Kit requires a smaller amount of primers and probes (e.g., 0.4 μM of each primer and 0.2 μM probe) than TaqMan Gene Expression Assays, the amount of assay in the duplex-reaction was reduced to determine the effect on the kit performance. HeLa and HepG2 cell-lines were used in RT-PCR duplex reactions with VIC-labeled GAPDH probes and FAM-labeled probes for the genes of interest.
A reduction in the concentration of TaqMan Gene Expression Assay, down to 0.5–0.6x, clearly improved the linearity of the assays (see figure
Determination of optimal assay concentration). The expected C
T differences of the dilution steps were determined using a 50% concentration of TaqMan Gene Expression Assay for LAMP3 and IFIT1. Reducing the assay concentration to 50% was critical for the detection of another gene of interest (GOI). For other targets and cell-lines, a concentration between 0.5x and 1x of TaqMan Gene Expression Assay was found to be optimal. In instances where 1x TaqMan Gene Expression Assays provided reasonable C
T values (for targets MAPK1, HERC5, ACTB, and GAPDH), a reduction in assay concentration had no effect.
Conclusions In this study, the tolerated stopping points and component storage conditions for the
FastLane Cell Multiplex Kit workflow were determined. Furthermore, a procedure enabling optimal results when using FastLane Cell Kits with TaqMan Gene Expression Assays was described. These results provide guidance in customizing the automated workflow procedure for optimal performance (see
Modified workflow based on study results).
