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Recovering RNA From Small Samples RNAqueous™-Micro
Kit
• Quantitative
RNA recovery from 4 - 400,000 cells
• Generates
concentrated RNA for immediate use in downstream applications
• Includes
DNA-free™ technology to remove contaminating
genomic DNA
Molecular analysis of gene expression
from very small samples can be challenging. The RNA isolation method
used for limited samples should provide for quantitative recovery
of RNA that is intact, free from contaminants, and as highly concentrated
as possible. Increasingly, expression analysis is being carried out
on "micro-sized" samples, including samples obtained by Laser Capture
Microdissection (LCM) and related microdissection techniques; needle
biopsies; fine dissection; and samples comprised of low numbers of
cultured cells. In these cases, it is desirable to recover the total
RNA in a small volume so that the entire sample can be used in downstream
applications, such as reverse transcription. Also, keeping the sample
as concentrated as possible facilitates analysis and quantitation
of the total RNA using sensitive microfluidic assays (e.g. Agilent
2100 bioanalyzer). Ambion's RNAqueous™-Micro Kit
was developed to optimize recovery of highly concentrated total RNA
from micro-sized samples.
Isolate Exceedingly Pure Total
RNA in a Small Volume
The RNAqueous-Micro Kit begins with sample
disruption in a chaotropic solution to lyse the cells and inactivate
cellular nucleases. The cell lysate is loaded onto a glass fiber
filter, washed and eluted. The filter cartridge provided in the
RNAqueous-Micro Kit is only a few millimeters in diameter so
that it can be saturated with a small volume of fluid. This allows
the RNA to be eluted in a volume of only 20 µl. After elution,
the RNA is treated with DNase I to eliminate genomic DNA contamination
that can interfere with RT-PCR assays. The DNase is then removed
using the DNA-free™
Removal Reagent which removes the DNase I and buffer ions
in a simple, quick step without organic extraction or heat inactivation.
(Heat inactivation of DNase frequently causes divalent cation-mediated
degradation of the RNA.) Using the RNAqueous-Micro Kit, the entire
RNA isolation procedure, including DNase treatment, takes about
30 minutes.
High Quality RNA for qRT-PCR
To demonstrate the quality of RNA isolated
by the RNAqueous-Micro Kit, RNA from 4 - 400,000 cells was recovered
using the procedure and analyzed for integrity and concentration.
Figure 1B shows an electropherogram of some of this total RNA.
The RNA was intact. The RNA was then used in a one step qRT-PCR
assay, and linear recovery of mRNA was shown over a range of 1 100,000
cell-equivalents (diluted from the 4 - 400,000 cells; Figure 1A).
Minus RT reactions showed no amplification (data not shown).
Figure 2 shows an electropherogram
and real-time PCR data generated from cells isolated by LCM and RNA
purified using the RNAqueous-Micro Kit. The RNAqueous-Micro Kit contains
sufficient reagents and filter cartridges to isolate total RNA free
of DNA from 50 samples of 1 - 1 x 105 cells or up to 5
mg of tissue.
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Figure 1. (A)
Linear Recovery of RNA from Cells Over a 5 Log Range. RNAqueous™-Micro
Kit was used to isolate total RNA from K562 cells diluted
from 100,000 cell equivalents down to 1 cell equivalent.
The RNA samples were then used for amplification of GAPDH
by real time RT-PCR. (B)Electropherogram
of RNA isolated from 10,000 K562 cells. RNA was run
on an Agilent 2100 bioanalyzer. Total yield after DNase
treatment and clean up was 360 ng with an 28S/18S rRNA
ratio of 1.87. |
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Figure 2. (A)
Use of LCM Samples for One Step RT-PCR of Neurogranin. RNA
was isolated from nine mouse brain Laser Capture Microdissection
samples using the RNAqueous™-Micro Kit.
The 20 µl post isolation eluates were DNase treated
(final volume 25 µl). A fifth of this treated sample
was subjected to one-step RT-PCR for analysis of neurogranin
(Ct of 18.44).
(B) Electropherogram
of RNA Isolated from the Granule Cell Layer of Hippocampus
(Mouse Brain). Cells were obtained through LCM
and RNA was isolated with the RNAqueous™-Micro
Kit. 5 µl was run and analyzed on an Agilent
2100 bioanalyzer. Total yield after DNase treatment
and clean up was 120 ng with an 28S/18S rRNA ratio
of 0.72. |
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RNA Isolation: The Basics
[read]
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The Do's and Don'ts of Total RNA Isolation
[read]
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Laser Capture Microdissection (LCM) is
a method for obtaining pure populations of cells from heterogeneous
samples. LCM permits the selection and capture of cells, cell aggregates,
and discrete morphological structures from thin tissue sections.
For LCM, frozen and OCT embedded, or formalin-fixed and paraffin
embedded tissue sections are processed, stained, and dehydrated,
then scanned under an inverted microscope. Cells are visualized through
a thermoplastic film which is attached to the bottom of an optically
clear microfuge tube cap. A laser pulse, directed onto the target
cells through the film, melts the film and allows it to flow onto
the targeted area where it cools and bonds with the underlying cell(s).
The film including the adhered cells or clusters is then lifted.
Captured cells can then be used for nucleic acid studies, including
SNP analysis, endpoint and real-time RT-PCR, and mRNA expression
profiling.
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