Navigation Links
New Kit Generates Exceptionally Pure Total RNA for RT-PCR


High-quality RNA for RT-PCR using the Absolutely RNA RT-PCR miniprep kit

Karen Dolter Jeff Braman
Stratagene

Stratagene has determined that standard RNA purification kits are not satisfactory for reverse transcribed PCR (RT-PCR) applications. We tested several commercially available RNA purification kits and have shown that contaminating DNA will produce false positive amplicons during RT-PCR. The Absolutely RNA RT-PCR miniprep kit eliminates contaminating DNA and produces highly pure total RNA qualified for RT-PCR reactions from 5 to 40 mg of tissue and 10 5 to 10 7 cultured cells. The Absolutely RNA RT-PCR miniprep kit utilizes a solid-phase DNase treatment to reduce DNA contamination to levels undetectable by PCR.

RT-PCR is an important technique for gene discovery and gene expression analysis because it amplifies expressed RNA sequences.1 RT-PCR is very sensitive; it facilitates the investigation of rare mRNA transcripts that are difficult to detect by other means in studies in which the quantity of starting material is limited. High yield of RNA is important because diminished or biased recovery may result in the loss of rare mRNA. Extreme purity is critical because contaminating DNA present in the RNA sample can give rise to amplification products that mimic the amplification product expected from the RNA target. The Absolutely RNA RT-PCR miniprep kit eliminates these problems by providing high yields of total RNA with undetectable levels of DNA from animal tissues and cultured cells.2

Eliminate DNA Contamination

Fig.1

In Figure 1, PCR analysis was performed to demonstrate the absence of DNA contamination in RNA isolated from different quantities of mouse tissue and cells. Using the Absolutely RNA kit, total RNA was isolated from mouse liver and mouse NIH/3T3 cells. From 5, 20, and 40 mg of liver, RNA yields were 14, 37, and 100 g, respectively, and from 105, 106, and 107 cells, RNA yields were 2, 19, and 142 g, respectively. Using Stratagenes prostar first strand RT-PCR kit3, cDNA was synthesized, and control reactions were run in the absence of reverse transcriptase. A 1-l aliquot of each of these reactions was used in PCR (Methods). The PCR included primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences that are present in greater than 200 copies in the mouse and rat genomes.4 The generation of a 0.2-kb PCR product from the control samples lacking reverse transcriptase (RT) indicates that DNA is present in the RNA sample. The absence of PCR product in the RT minus samples demonstrates that DNA is not detectable in the RNA purified with the Absolutely RNA kit.

RNA Samples Isolated Using Various Kits

Total RNA was isolated from tissue and cultured cells using several commercial kits, then examined by RT-PCR to determine which samples contained DNA contamination. RNA was isolated using Ambions RNAqueous kit, Clontechs NucleoSpin RNA kit, Qiagens RNeasy kit and Stratagenes Absolutely RNA RT-PCR miniprep kit, according to the instructions provided for each. An additional kit (Boehringer Mannheims High Pure RNA Isolation k it) was omitted from this comparison because it is not suited for tissue samples and is limited to RNA isolation from no more than 106 cells.

Stratagenes ProSTAR first strand RT-PCR kit was used to synthesize cDNA3 and control reactions were run in the absence of reverse transcriptase. An aliquot of each of these reactions was used in PCR (Methods). RNA isolated using the kits from Ambion, Clontech, and Qiagen contained DNA contamination, as shown by the presence of a 0.2-kb PCR band in the control lanes (Figure 2). In contrast, the RNA isolated using the Absolutely RNA kit shows no evidence of DNA. This result demonstrates that the Absolutely RNA miniprep kit is effective at eliminating DNA contamination and competitive kits are not.

Fig.2

Accurate Representation

Recovery of RNA from all abundance classes is important for accurate assessment of gene transcription levels. In addition, preservation of the distribution and abundance of mRNAs is important for generating optimal cDNA libraries. RT-PCR was used to assess the presence of low-, medium-, and high-abundance classes of mRNA (corresponding to approximately 0.1% or less, 0.1 to 1%, and 1% of total RNA, respectively)5 in total RNA isolated from mouse liver and testis using the Absolutely RNA kit.

For the experiment shown in Figure 3, the ProSTAR first strand RT-PCR kit was used to synthesize cDNA from the RNA.3 The resulting cDNA was used in PCR with Stratagenes control RT-PCR primers for genes of varying abundance levels in the mouse.5,6 These include primers for high-abundance transcripts (mouse elongat ion factor 1a), medium-abundance transcripts (b2-microglobulin), and low-abundance transcripts (protein phosphatase 1 and ornithine decarboxylase). Although b2-microglobulin mRNA is classified as medium abundance, its expression level is higher in liver than in brain7 (Figure 3). Similarly, ornithine decarboxylase is expressed at overall low levels but the levels vary in tissues throughout the body.8 The presence of the expected PCR bands in Figure 3 shows that even rare RNA transcripts are represented in total RNA isolated with the Absolutely RNA kit.

Conclusions

RNA isolated with Stratagenes Absolutely RNA RT-PCR miniprep kit is ideal for RT-PCR. The kits quick, convenient protocol includes a DNA removal step that ensures sensitive RT-PCR results without having amplification products generated from genomic DNA targets. Moreover, RT-PCR readily detects low-abundance transcripts from this RNA. The Absolutely RNA RT-PCR miniprep kit effectively generates high-purity, intact total RNA from a range of tissues and cultured cells, ready for the most sensitive RT-PCR experiments.2

Methods

The ProSTAR first strand RT-PCR kit was used for cDNA synthesis.3 For GAPDH primer detection of DNA in total RNA samples, control cDNA synthesis reactions were performed in the absence of reverse transcriptase. PCR reactions contained 1 l from the cDNA reactions (100 ng of total RNA), 200 M of each dNTP, 25 ng of each GAPDH primer, 1X Taq polymerase buffer, and 1.25 U of Taq2000 DNA polymerase. PCR react ions were performed in a RoboCycler 96 temperature cycler, starting with a 1-minute incubation at 95C and followed by 30 cycles of 95C for 30 seconds, 55C for 1 minute, and 72C for 30 seconds.

For PCR to detect transcripts of varying abundance levels, Taq2000 DNA polymerase was used according to instructions provided with the primers. For each primer set in the PCR reactions, 1 l of cDNA was used.

REFERENCES
Larrick, J.W. (1992) Trends Biotechnol. 10: 146-152.
Dolter, K. and Braman, J. (2000) Strategies 13: 12-14.
Mackman, C. H., Kubitz, M., and Willbanks, A. (1990) Strategies 3: 26-27.
Piechaczyk, M., et al. (1984) Nature 312: 469-471.
Bishop, J. O., et al. (1974) Nature 250: 199-204.
Adams, M. D., et al. (1995) Nature 377(6547S): 3-174.
Drezen, J. M., Babinet, C., and Morello, D. (1993) J. Immunol. 150: 2805-2813.
Wen, L., Huang, J.K., and Blackshear, P.J. (1989) J. Biol. Chem. 264: 9016-9021.

'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications
2. High-Throughput Isolation of Total RNA
3. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Total RNA Isolation Protocol 1,2,3
6. Isolate It All: siRNA miRNA Total RNA Native Protein
7. Isolate Total RNA and Protein From the Same Sample
8. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
9. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
10. Total RNA from Whole Blood for Expression Profiling
11. RNAPrep - Total RNA Isolation Purification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/27/2017)... ... March 27, 2017 , ... A research team led by ... in JoVE’s Video Journal, the world’s first peer-reviewed scientific video journal. The article ... coronary artery disease (CAD). Lam is an assistant professor at the Department of ...
(Date:3/27/2017)... DarioHealth Corp. (NASDAQ: DRIO), a leading global ... solutions, today announced that it is now offering a ... consumers who want to have their DarioHealth products reimbursed ... alliance agreements with partners across the U.S. who will ... approved, will supply and bill the customer,s insurance for ...
(Date:3/24/2017)... , March 24, 2017 MiMedx Group, ... company utilizing human placental tissue allografts and patent-protected processes ... multiple sectors of healthcare, announced today  that it will ... New York , NY.  Parker H. "Pete" Petit, ... Financial Officer, Christopher M. Cashman , EVP and ...
(Date:3/24/2017)... Research and Markets has announced the addition ... Strategic Business Report" report to their offering. ... This report analyzes the Global ... Annual estimates and forecasts are provided for the period 2014 through ... research. The report profiles 25 companies including many ...
Breaking Biology Technology:
(Date:2/28/2017)... --  Acuant , a leading provider of data capture ... new and core technologies building upon the acquisition of ... desktop Acuant FRM TM facial recognition and match ... manual review of identity documents by accredited professionals. ... most accurate capture software to streamline workflows by securely ...
(Date:2/24/2017)... Feb. 24, 2017  EyeLock LLC, a leader of iris-based ... iris biometric solution on the latest Qualcomm® Snapdragon™ ... Mobile World Congress 2017 (February 27 – ... Hall 3, Stand 3E10. The ... security platform—a combination of hardware, software and ...
(Date:2/14/2017)... -- Wake Forest Baptist Medical Center today announced Julie Ann Freischlag, ... (CEO). Freischlag joins the medical center on May 1 ... who last year announced that he would transition to ... it since 2008.   As CEO, Freischlag ... academic health system, which includes Wake Forest School of ...
Breaking Biology News(10 mins):