Sample Preparation |
Purification of Total RNA
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Applications
- Purification of RNA from cultured cells for microarray analysis or real-time
RT-PCR.
The ArrayPure™ Nano-scale RNA Purification Kit provides all of the reagents
needed to purify RNA from one to a few thousand eukaryotic cells. The reagents
are all aqueous to avoid the use of toxic organic solvents.1 The
nano-scale protocol has been developed and tested with quantitative real-time
PCR on 1 to 10,000 eukaryotic cells (Figs. 1-3). In addition, microgram amounts
of RNA have been produced from 20 HeLa cells using aRNA two-round synthesis
techniques. The kit includes ScriptGuard™ RNase Inhibitor to maintain
the integrity of purified RNA.
Benefits
- Purify RNA from as little as one cell.
- Simple procedure with no toxic solvents.
- Captures low- and high-molecular-weight RNA.
- Does not use columns.
References
- Miller, S.A. et al. (1988) Nucl. Acids Res. 16, 1215.
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Figure 1. Quantitative real-time PCR extension profiles of RNA purified
using the ArrayPure™ Nano-scale RNA Purification Kit. Intact HeLa
cells were serially diluted ten-fold in growth medium to dispense 104, 103,
100, 10 or 0 (medium control) cells per tube. (Note: these were actual dilutions
of living cells and not dilutions of a cell lysate, as some commercial vendors
have done.) The RNA in each tube was purified with the ArrayPure Kit. The
purified HeLa RNA was converted to cDNA using EPICENTRE's MMLV Reverse Transcriptase.
These corresponding cDNAs were amplified by the FailSafe™ Real-Time
PCR System on a Bio-Rad iCycler iQ™ Real-Time PCR Detection System. |
Figure 2. Linear Plot of CT vs Log of Initial HeLa Cell Number. The
log of initial cell number for the amplified cDNA made from the RNA purified
in Fig. 1 was plotted against cycle threshold (CT) values. Slope:
-3.75; r2 = 0.991. The CT linearity extends for four orders
of magnitude of initial cell number.
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Figure 3. Quantitative real-time PCR amplification plot. HeLa cells
were grown in tissue culture, aseptically diluted, and trapped inside sterile
5-µl microcapillary pipets. The number of cells isolated was verified
by observation with an inverted microscope. Then, the cells were eluted by
centrifugation from the capillary pipet, washed with phosphate-buffered saline,
and the RNA was purified by the ArrayPure™ Nano-scale RNA Purification
Kit. Purified HeLa RNA was converted to cDNA using EPICENTRE's MMLV Reverse
Transcriptase. The corresponding cDNAs were amplified using the FailSafe™ PROBES
Real-Time PCR PreMix Choice Kit (PreMix 3). |
Figure 4. CT vs Log of Initial HeLa Cell Number is Linear. The
log of the initial cell number for the amplified cDNA made from the RNA purified
from an average of 100, 10 , and 1 HeLa cell was plotted against cycle threshold
(CT) values. Slope = -3.14; r2 = 0.992. The CT linearity
extends for 3 orders of magnitude of initial cell number.
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| Figure 5. Melt Curve Analysis of the Quantitative Real-Time
PCR. The cDNA corresponding to RNA purified from 10, 100, 103 and
104 HeLa cells yielded PCR products (peaks at 87°C to 88°C).
The 0 cell (medium control; black line) sample yielded only primer dimers
(peak at 80°C to 81°C) indicating the absence of detectable RNA,
as expected. |
Figure 6. HeLa cell RNA was purified from two separate
tissue culture flasks, labeled with Cy™3 or Cy™5, and hybridized
to a microarray containing Operon's Array-Ready Human Oligo (70-mer) Set™. |
FailSafe™ GREEN and PROBES, TAQurate™ GREEN and PROBES, MasterAmp™ GREEN, TAQXpedite™ GREEN (discontinued)
MasterAmp™ RT-PCR Kit for High Sensitivity
MessageBOOSTER™ Whole-Transcriptome cDNA Synthesis Kit for qPCR
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