Navigation Links
Simple Isolation of RNA from Tissue and Cultured Cells


Easy RNA purification with integrated DNA-removal step

Karen Dolter Jeff Braman
Stratagene

The StrataPrep total RNA miniprep kit efficiently isolates high-quality total RNA from 10 5 to 10 7 cultured cells or 10 to 40 mg of tissue and includes an on-column DNA-removal step. Additionally, multiple samples can be processed simultaneously because of the easy-to-follow technique. The RNA isolation is performed within microspin cups, which eliminates the need for organic extraction or ethanol precipitation. The resulting total RNA is suitable for a wide variety of demanding molecular biology applications.

Fig.1

High-quality RNA is necessary for techniques such as cDNA synthesis, RT-PCR amplification, RNase protection assay, primer extension analysis, and Northern blotting. Stratagenes StrataPrep total RNA miniprep method takes advantage of the efficient denaturing properties of guanidine thiocyanate for cell lysis. The protein-denaturing ability of this chaotropic salt facilitates the isolation of intact RNA from tissue rich in ribonucleases. The method originally described1 has been simplified by using a silica-based fiber matrix in a microspin-cup format (Figure 1). The RNA is immobilized on the fiber matrix, allowing contaminant removal while avoiding organic extractions. In addition, no ethanol precipitation of the RNA is necessary. By treating the RNA sample directly on the fiber matrix with DNase, DNA contamination is reduced to undetectable levels. Once the purified RNA is eluted from the fiber matrix in a small volume of buffer or water, it is ready to use.

We showed that the kit is capable of high performance using a wide range of starting materials, both tissues and cells. We then assessed the quality of the isolated RNA by absorbance measurements, gel electrophoresis, RT-PCR, and molecular beacon real-time RT-PCR.

RNA Isolation from Different Quantities and Sources of Tissue and Cells

Table 1
RNA Yield and Purity from Varying Amounts of Rat Liver

RNA was isolated from the specified amounts of tissue using the StrataPrep total RNA miniprep kit. Yield and purity were determined by measuring absorbance in 10 mM Tris, pH 7.5.

Amount of rat liver

Average yield (g)

Average A260/A280

Number ofisolations

5 mg

14 0.8

2.1 0.07

3

10 mg

26 10.1

2.1 0.06

7

20 mg

51 14.1

2.0 0.09

11

30 mg

94 11.4

2.0 0.02

6

40 mg

122 15.4

2.0 0.04

10

Table 1 includes yield and purity data for RNA isolated from varying amounts of rat liver, using the kit. The RNA yields were good, averaging 14 to 122 g for liver quantities between 5 and 40 mg; individual yields reached as high as 141 g. The purity, as determined by the A260/A280 ratio, was 1.9 or greater (Table 1).

Table 2
Isolation of RNA from Different Cell Lines

RNA was isolated from 2 x 106 cells using the StrataPrep total RNA miniprep kit. Yield and purity were determined by measuring absorbance in 10 mM Tris, pH 7.5.

Species

Cell line

Average yield (mg/2 x 106 cells)

Average A260/A280

Number of
isolations

Mouse

NIH/3T3

41 6.2

2.1 0.07

8

Hamster

CHO

25.0 0.75

2.2 0.04

3

Human

HL-60

13.1 0.62

2.1 0.01

3

Table 2 and Table 3 show yield and purity of RNA isolated from a variety of cells and tissues. The RNA exhibited A260/A280 ratios of greater than 1.9 for each sample, indicating high purity of the RNA. Approximately 1 g of representative RNA samples was electrophoresed on formaldehyde-agarose gels to assess the integrity of the RNA. Figure 2 shows intact 28S and 18S ribosomal RNA bands for each sample.

Fig.2

Table 3
Isolation of RNA from Various Mouse Tissues

RNA was isolated from different amounts of mouse tissues using the StrataPrep total RNA miniprep kit. Yield and purity were determined by measuring absorbance in 10 mM Tris, pH 7.5.

Tissue

Average yield (mg/mg tissue)

Average A260/A280

Number of isolations

Brain

0.7 0.14

2.2 0.12

4

Heart

0.3 0.06

2.2 0.21

2

Kidney

2.0 0.22

2.2 0.11

4

Liver

4.2 1.05

2.1 0.02

4

Lung

1.2 0.15

2.1 0.04

2

Spleen

3.6 0.88

2.2 0.19

4

Testis

1.6 0.36

2.2 0.14

4

Assay for DNA Contamination

With the RT-PCR technique, small quantities of genomic DNA contamination can be detected in RNA samples. This method was used to demonstrate the effective removal of DNA using the StrataPrep total RNA miniprep kit (Figure 3). Primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences (present in approximately 200 copies in mouse and rat genomes2) were used, providing an especially sensitive assay for the presence of DNA. PCR amplification of GAPDH sequences from cDNA shows the expected 0.2-kb fragment (Figure 3: Lanes 1, 3, 5, and 7). When the DNase treatment step is omitted from the protocol and reverse transcriptase is omitted from the cDNA synthesis reaction, the presence of DNA is detected in the PCR (Figure 3: Lanes 4 and 8). However, when the StrataPrep total RNA miniprep protocol is carried out according to the kits instructions, no DNA contamination is detected (Figure 3: Lanes 2 and 6).

Fig.3

Detecting RNA of Varying Abundance

RNA isolated with the StrataPrep total RNA miniprep kit was used to synthesize cDNA, which served as a template for amplification using Stratagenes RT-PCR Primers for Human Gene Transcripts of Varying Abundance Levels,3 according to the instructions provided. The presence of PCR products for high- (-actin), medium- (g-actin) and low- (protein phosphatase 1, ADP ribosylation factors 1 and 3, and ornithine decarboxylase) abundance mRNA shows that a wide spectrum of RNA molecules is present in the sample (Figure 4).

Fig.4

Sensitive Detection of RNA Using Molecular Beacons

Molecular beacon technology provides an elegant and efficient method for PCR and RT-PCR that does not require gel electrophoresis of the products and permits accurate quantitation.4 The Mx4000 molecular beacon expression analysis kit for mouse -actin5 was used for real-time RT-PCR analysis of RNA isolated from NIH/3T3 cells with the StrataPrep total RNA miniprep kit. RNA is readily detected from a sample as small as 1 pg in a target titration of 1 100,000 pg of total RNA (Figure 5). These results demonstrate that the RNA isolated using the StrataPrep kit is of the high quality required for real-time RT-PCR.

Fig.5

Other Spin-Cup-Based Total RNA Isolation Kits

Most spin-cup-based total RNA isolation kits from other manufacturers do not include a DNase digestion step in the standard protocol. When these kits are used for samples that require DNA removal, DNase treatment must be performed after isolating the RNA, which is then followed by repurifying the RNA to remove the DNase. Extra materials are not provided in these kits to perform these steps. In some kits that include DNase digestion within the protocol, only very small quantities of tissue and/or cells can be used. Other RNA isolation kits that provide DNase require extra steps, such as heating and a long centrifugation or repurification of the RNA.

Conclusions

The StrataPrep total RNA miniprep kit is a quick and convenient method for isolating pure total RNA with high yields and no evidence of DNA contamination from a variety of cells and tissue. Use the purified RNA in cDNA synthesis, RT-PCR amplification, RNase protection assay, primer extension analysis, Northern blotting, and other applications. Additionally, follow this procedure to purify RNA after enzyme reactions (to remove RNA polymerase, DNase, etc.).

Methods

To isolate RNA, tissue or cultured cells are homogenized in lysis buffer, then the sample is passed through a prefilter by centrifugation. The prefilter removes particulates and much of the DNA contamination. The clarified homogenate is mixed with ethanol and applied to the fiber matrix within the microspin cup for binding of the RNA. The RNA is washed, then the sample is digested with DNase directly on the fiber matrix. Additional washing of the microspin cup removes the DNase and impurities, and the RNA is eluted from the fiber matrix with a low ionic strength buffer.

Acknowledgments

We thank Mark Dycaico for rat and mouse tissues, Michelle Cayouette for GAPDH primers and Gothami Padmabandu and Reinhold Mueller for molecular beacon RT-PCR analysis.

REFERENCES

  1. Chirgwin, J. M., et al. (1979) Biochemistry 18: 5294-5299.

  2. Piechaczyk, M., et al. (1984) Nature 312: 469-471.

  3. Rogers, B. and McKenzie, D. (1998) Strategies 11: 100-102.

  4. Cayouette, M., et al. (1999) Strategies 12: 85-88.

  5. Padmabandu, G. and Mueller, R. (1999) Strategies 12: 94-97.


'"/>

Source:


Page: All 1 2 3 4 5 6 7 8

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
3. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
4. Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies
5. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
6. Simple and Rapid Isolation of Plant RNA Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit
7. The Fastest, Simplest, and Most Effective Way to Remove DNA Contamination
8. Simple Approaches for Optimization of RT-PCR
9. Detection of Morphine and Morphine-Glucuronide in Saliva or Urine with a Rapid and Simple Method Without Derivatization on the Varian 500-MS
10. Gram Quantities of MAb Produced with Simple Bioreactor in Serum-Free Perfusion Culture Replacing Ascitic Fluid Production
11. Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Simple Isolation RNA from Tissue and Cultured Cells

(Date:7/31/2015)... July 31, 2015 In today,s fast ... help them drive positive health outcomes, improve profitability ... of patient care in their communities. At ThoughtSpot ... and conference, AmerisourceBergen announced a new set of ... Administration Organization and help independent pharmacies endure the ...
(Date:7/30/2015)... ... July 31, 2015 , ... Ralco is honored to announce that ... County Fair to be held August 5-9 in Marshall. The Ralco Enrichment Center is ... from and how agriculture impacts their daily lives. This unique exhibit also features a ...
(Date:7/30/2015)... ... 30, 2015 , ... The 2015 Market Research Report on the Global ... the global Propanol market with a focus on the Chinese situation. Major companies included ... Oxea, ISU Chemical, Sanofi, Royal Dutch Shell, LG Chem and Zhejiang Xinhua Chemical. , ...
(Date:7/30/2015)... 30, 2015   Senomyx , Inc. (NASDAQ: ... technologies to discover, develop, and commercialize novel flavor ingredients ... reported financial results for the second quarter 2015. ... we remain on track to achieve our commercial and ... Officer of the Company. "Since our last quarterly earnings ...
Breaking Biology Technology:AmerisourceBergen Unveils Suite of Enhanced Capabilities for Independent Pharmacies at ThoughtSpot 2015 2AmerisourceBergen Unveils Suite of Enhanced Capabilities for Independent Pharmacies at ThoughtSpot 2015 3Ralco Announces Primary Sponsorship of Ralco Enrichment Center at Lyon County Fair 2Global Propanol Market Technical Data and Manufacturing Analysis 2020 2Global Propanol Market Technical Data and Manufacturing Analysis 2020 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 2SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 4SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 5SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 6SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 7SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 8SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 9
... prior to the 1984 divestiture, the prime directive in providing ... will provide universal service to everyone". , ,In exchange ... phone companies ensured that everyone got service to their house ... across the board. That worked very well and the network ...
... Congress has its way, English will become the official language of ... soccer tournament beat them to it. , ,For the first ... proficiency in written and spoken English in order to be among ... If any game is international, its football, as its called in ...
... may not agree, Memorial Day marks the traditional start of ... the three-day weekend to return to work, sure in the ... are around the corner, and the next three months will ... and barbeque. , ,It's the summer slide, when late nights ...
Cached Biology Technology:Does redefining universal service help bridge the digital divide? 2Does redefining universal service help bridge the digital divide? 3Does redefining universal service help bridge the digital divide? 4Does redefining universal service help bridge the digital divide? 5English as the lingua franca of a new age: Its more powerful than any law 2English as the lingua franca of a new age: Its more powerful than any law 3Summer marks countdown to DEMOfall 2Summer marks countdown to DEMOfall 3
(Date:7/21/2015)... -- Today, ZTE announced its Android smartphone Axon, featuring ... revenues in 2015 that relate to sales of FPC1025 for this ... 2,200 MSEK for 2015. Jörgen Lantto, CEO of FPC, ... China and we are proud that ... Axon , its first ...
(Date:7/13/2015)... , July 13, 2015 Synaptics Inc. ... human interface solutions, today announced sampling of ClearPad ... display driver integration (TDDI) product targeting smartphones and ... to combine Synaptics , best-in-class touch controller ... driver technology developed in the company,s Japan Design ...
(Date:7/9/2015)... 2015  Unchained Labs just can,t get enough ... Avid Nano. Avid Nano designs, develops and manufactures ... Also today, Unchained Labs UNcaged the ... use protein sizing system. The pUNk is a ... size, size distribution, aggregation population and molecular weight. ...
Breaking Biology News(10 mins):FPC's Touch Fingerprint Sensor FPC1025 in ZTE's Smartphone Axon 2Synaptics Announces Sampling of Second Generation TDDI Solutions 2Synaptics Announces Sampling of Second Generation TDDI Solutions 3Unchained Labs just can't get enough, acquires Avid Nano, rocks out the new pUNk! 2
... each year and, according to the World Health Organisation, in ... spread by mosquitoes which breed in open water and spend ... at the water surface. New research published in BioMed Central,s ... method of dispersing pathogenic fungi as a means of preventing ...
... Mich. Ask any political leader if they are ... typically a resounding, "Yes." Evaluating its effectiveness, however, ... and environmental science and policy professor at Michigan State ... evaluate sustainability at a national gathering of scientists Feb. ...
... A defining feature of a scientific discovery is ... this means higher standards of communication of discoveries ... with the published research paper. Doing this makes the ... verification of results. Tools and approaches to facilitate ...
Cached Biology News:It's time to take a unified approach toward measuring sustainability 2AAAS Symposium: New research facilitates scientific knowledge transfer 2
Biotin-OSu is used to label proteins, immunological agents, DNA and synthetic oligonucleotides. Biotin labeling is detected through enzymatically labeled avidin or streptavidin proteins that conforma...
Cross reactivity to St. Louis encephalitis (MSI-7) is determined by ELISA....
ANTI K. AEROGENES...
casein kinase 2, alpha 1 polypeptide, transcript variant 2, mRNA (cDNA clone MGC:15232 IMAGE:4121882), complete cds...
Biology Products: