Description

MagExtractor-RNA kit provides a simple and reliable method for the rapid purification of total RNA from various specimens (e.g. cultured cells or animal tissues) using magnetic silica beads. This kit is based on binding properties of RNA onto a silica surface in the presence of chaotropic agents (1)(2) and an RNA-binding accelerator. Purified total RNA contains primarily rRNA and mRNA. The purified total RNA can be used for RT-PCR experiments.

Features

  • Does not contain hazardous substances, such as phenol or chloroform.
  • No ethanol is used in the washing steps.
  • Suitable for high-throughput extraction of total RNA from various specimens.

Applications

  • Extraction of total RNA from cultured cells, animal tissue and yeast cells.
Sample* Amount of specimens Yield Remarks
Cultured cells ~5x106 cells ~10 µg/106 cells Total RNA yields depend on the number of cells.
Tissue ~30 mg ~15 µg/30mg Total RNA yields depend on tissues or storage conditions.
Yeast ~5 O.D. (660nm)/ ml ~20 µg/5 O.D. Pretreatment by zymolyase is necessary.

*This kit is not applicable for extraction from whole blood or serum. In the case of whole blood, white blood cells should be separated by a centrifugation using Ficoll.

Storage condition

Store at 4ºC

Components

Lysis & Binding Solution* 77 ml
Washing Solution I 66 ml
Washing Solution II 176 ml
Elution Solution 10 ml
Magnetic Beads 6 ml

*1/100 volume of 2-mercaptoethanol must be added prior to use.

Principle

The selectivity of extracted nucleic acids can be changed by optimization of the binding and washing solutions. MagExtractor -Genome- (Code No. NPK-101) extracts genomic DNA from various specimens (e.g. whole blood, cultured cells or animal tissues etc.). MagExtractor -RNA- (Code No. NPK-201) extracts total RNA from various specimens (e.g. cultured cells or animal tissues). MagExtractor -Plasmid- (Code No. NPK-301) extracts plasmids from E. coli cells, MagExtractor -Viral RNA- (Code No. NPK-401) is a kit for extracting viral RNA from serum or plasma specimens. MagExtractor -Plant Genome- (Code No. NPK-501) is a kit for extracting genomic DNA from various plant specimens (e.g., leaf, cultured cells, etc.). MagExtractor-PCR & Gel Clean up- (Code No. NPK-601) extracts DNA fragments from a PCR solution, enzyme solution, or agarose gel slices.

Application data

Example 1. Purification of total RNA from cultured cells

Total RNA were purified from HeLa cells (2x106 cells) using MagExtractor -RNA- and a spin column kit (company A). Then, the transferrin receptor gene (2115 bp) was amplified using the purified RNA by RT-PCR. As shown in Fig. 1 and Table 1, MagExtractor -RNA- showed excellent RNA yields, comparable with the spin column kit. Distinct amplified bands were obtained by RT-PCR using total RNA from MagExtractor -RNA- as well as the spin column kit (Fig.2).

Fig. 1. Electrophoretic analysis of the purified total RNA

1: MagExtractor
2:Company A
(spin column)


Fig. 2. Analysis of the amplified products by RT-PCR

Sample:
1: RNA from MagExtractor Kit
2: RNA from Company A

Table 1. Comparison of yields and purity

  Yield
(A260 nm)
Yield (A260 nm)
A260/
280 nm
A260/280 nm
MagExtractor 27.1 µg 2.0
Company A (Spin column) 27.3 µg 2.0

Example 2. Purification of total RNA from rat tissues

Total RNA were purified from various rat tissues (50-100 µg) using MagExtractor -RNA-, showing excellent RNA yields (Fig.1, Table 1).

Fig. 1. Electrophoretic analysis of purified total RNA

1: Liver
2: Testis
3: Brain
4: Kidney

Table 1. Comparison of yields and purity

  Yield
(A260 nm)
Yield (A260 nm)
A260/
280 nm
A260/280 nm
Liver 10.4 mg 2.08
Testis 9.3 mg 2.09
Brain 5.2 mg 2.04
Kidney 9.7 mg 2.09

Example 3. Northern blotting using purified total RNA from cultured cells

Total purified RNA from HeLa cells was analyzed by Northern blotting using a probe prepared from the transferrin receptor gene (4.5 kb). As shown in Fig.2, distinct mRNA bands were detected of the expected size.

Fig. 1. Electrophoretic analysis (denaturing conditions)

M:λ/ Hind III Marker
1: RNA 10 µg
2: RNA 5 µg
3: RNA 2.5 µg


Fig. 1. Northern blotting analysis

1: RNA 10 µg
2: RNA 5 µg
3: RNA 2.5 µg

References

  • B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. USA. 76: 615-619 (1979)
  • R. Boom, C. J. A. Sol, M. M. M.Salimans, C. L. Wertheim-van Dillen, P. M. E. Dillen and J. van der Noordaa, J. Clin. Microbiol., 28: 495-503 (1990)