RNA Guide: Purifying RNA and mRNA - Promega

Purifying RNA and mRNA
Mid-scale, Single-prep Total
RNA Isolation
As standard molecular biology applications become more
sensitive, it is increasingly important for RNA isolations
to be free of contaminating genomic DNA. The
PureYield RNA Midiprep System uses a novel combination
of reagents, membrane, and protocol to achieve
pure RNA with undetectable genomic DNA contamination.
The isolation is completed without the use of a
DNase treatment, organic solvents, protease digestions,
or alcohol precipitations. The eluted RNA is ready for
sensitive downstream applications such as quantitative
RT-PCR, RT-PCR, and microarray analysis.
Amplification Curves
No detectable
gDNA by qPCR
1. Add isopropanol to the
cleared lysate.
2. Mix and transfer to
PureYield Binding Column.
1. Prepare lysate.
2. Add RNA Dilution Buffer. Mix.
3. Add Clearing Agent. Mix and vortex.
4. Incubate at 70°C for 5 minutes.
5. Cool for 5 minutes.
1. Transfer mixture to PureYield
Clearing Column in collection tube.
2. Centrifuge to clear the lysate.
No DNase
treatment
required
RFU
-1.1E5 -1.0E5 -9.0E5 -8.0E5 -7.0E5 -6.0E5 -5.0E5 -4.0E5 -3.0E5 -2.0E5 -1.0E5 0
DNA Standards
Competitor I
No-Template Control
PureYield
RNA Midiprep
5 10 15 20 25 30 35 40
Cycle
RNA purified with the PureYield RNA Midiprep System has no detectable
genomic DNA contamination. RNA was purified from 1 × 10 8 HEK293T using the
PureYield system and competitor “I’s” solution-based purification method. 100ng of
purified total RNA was analyzed using the Plexor qPCR System to determine the
quantity of gDNA using primers specific for the human thyroid peroxidase gene
(TPOX). Human Genomic DNA (Cat.# G3051) in quantites of 10 4 , 10 3 , 10 2 and
10 1 copies was used as a standard.
PureYield RNA Midiprep
System
Protocol available at:
www.promega.com/tbs/tm279/tm
279.html
Cat. # Z3740 and Z3741
5360TA
Centrifuge (Spin)
1. Centrifuge.
2. Wash 2X.
3. Centrifuge
10 minutes
to dry.
Vacuum (Vac)
1. Apply vacuum.
2. Wash 2X.
3. Dry 3 minutes.
1. Transfer PureYield Binding
Column to 50ml collection tube.
2. Add Nuclease-Free Water
and incubate 2 minutes.
3. Centrifuge 3 minutes to elute
pure RNA.
Schematic representation of the PureYield Total RNA Isolation System.
Average Yields of Total RNA Isolated from Tissues and Cells.
Sample
Sample Amount / Maxium Average Yield Average Average
Type Sample Capacity per Prep (µg) 1 A 260 /A 230 A 260 /A 280
Rat Tissues
Liver 300mg 990.7 1.8 1.9
Lung 300mg 193.9 2.0 2.1
Kidney 200mg 329.1 2.3 2.1
Spleen 150mg 430.9 2.3 2.1
Brain 300mg 305.5 2.3 2.1
Heart 300mg 255.3 2.2 2.1
Muscle 300mg 115.1 2.1 2.1
Bacteria
E. coli 1 × 10 10 cells 872.7 2.5 2.1
Plant Tissue
Canola 300mg 87.8 1.5 2.1
Cell Lines
HEK 293T 5 × 10 7 cells 453.3 2.1 1.9
HeLa 5 × 10 7 cells 329.2 1.8 2.0
Blood 20ml (10ml/tube) ~10* * *
1. The values represent means of results achieved at Promega. Yields will depend on the metabolic state of
the sample, culture conditions, harvesting conditions and sample preparations. The average total RNA yield
shown is from a 1ml elution.
* Varies by white cell count. A white cell count of ~5 × 10 6 cells/ml yields ~10µg of total RNA.
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