We developed a cell
lysis buffer that immediately stabilizes nucleic acids for use in
Quantitative Reverse Transcription-PCR (QRT-PCR) without further
processing. Using lysate dilutions as templates, the generated signal is
equivalent to that of purified nucleic acids; RNA in the lysate is stable
for storage up to six months at –20 ° C.
Introduction
The isolation of RNA from multiple samples is tedious, time consuming,
and often results in variable yields. SideStep™ Lysis and Stabilization
Buffer allows access to the entire nucleic acid complement by QPCR without
isolation procedures that can bias gene expression analysis.
Experimental conditions
Figure 1. QRT-PCR amplification plot
shows equivalent performance between serial dilutions of purified
mRNA and SideStep™ cell lysate.
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HeLa cells, grown in DMEM, were
harvested with trypsin and resuspended in DMEM containing 10% FBS. Cells
were washed in cold PBS and counted in a Coulter counter. Cells (1 x 105)
were pelleted in a 1.5-mL tube. The supernatant was removed and 100 μL
SideStep Lysis and Stabilization Buffer were added and the suspension was
vortexed for 1 min. Ten-fold serial dilutions of the lysate in water were
used as templates in our Brilliant® QRT-PCR Master Mix 1-step kit,
combined with an RNA-specific probe for GAPDH, and run on our Mx3000P®
QPCR System. Cycling parameters were 30 min at 50°C, 10 min at 95°C, 50
cycles of 15 s at 95°C and 1 min at 60°C. Similar reactions used mRNA
purified with the Absolutely mRNA™ Purification kit from the same number
of cells used for the lysate template. Control reactions were also run to
ensure sole amplification of cDNA. To demonstrate the stabilization of
RNA, a SideStep lysate of HeLa cells was stored at –20°C for 6 months
prior to isolating RNA using a column-based purification method. The
quality of the isolated RNA was assessed using the 2100 Bioanalyzer and
Pico RNA 6000 kit (Agilent).
Results
Figure 2. Electropherogram and RIN
data from Agilent 2100, showing the quality of HeLa RNA from a
SideStep™ cell lysate stored for 6 months at –20°C.
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Figure 1 shows the gene expression
signal from the SideStep lysate. One can see that this signal is equal to
that of isolated mRNA from the same number of HeLa cells. Figure 2 shows
the quality of RNA from the cell lysate after 6 months of storage at –20°C.
Further analysis yields a 28S:18S ratio of 1.9 and an RNA integrity number
of 8.5, proving that high-quality RNA is maintained in the cell lysate.
Conclusions
The SideStep Lysis and Stabilization
Buffer ensures the generation of accurate gene expression data by
minimizing the loss or degradation of nucleic acid molecules by skipping
the RNA and DNA purification steps and moving directly from cells to
QRT-PCR amplification. This buffer reagent allows the processing of
multiple samples, minimizes the variability in RNA recovery, and improves
the overall reliability of QPCR data.
