Navigation Links
Quantitation of RNA for Hybridization Blots Using the VersaFluor Fluorometer

Matthew W. Fields, Section of Microbiology, Cornell University, Ithaca, New York 14853


Introduction
Biologists frequently monitor the effect of environmental stimuli on living organisms by quantitating mRNA levels. The relative abundance of mRNA is typically determined with probes and hybridization blots, but these methods depend on accurate and sensitive methods for measuring total RNA. When fluorochromes bind to nucleic acids, fluorescence occurs, and changes can be monitored with a fluorometer. The following section describes the quantitation of total RNA with a VersaFluor fluorometer (Bio-Rad).


Materials and Methods
Total RNA was isolated from the strictly anaerobic ruminal bacterium, Prevotella bryantii, using an acid-phenol extraction procedure.1 All solutions, tubes, and pipets were DEPC treated and autoclaved before use. Escherichia coli K12 total RNA was isolated using a commercially available RNA purification kit and was stored at -70 C. RNA concentrations were determined using the VersaFluor fluorometer and RiboGreenTM RNA quantification kit (Molecular Probes, Inc). The excitation was at 490 nm (EX 490/10 filter), and the emission was monitored at 520 nm (EM 520/10 filter). A standard curve was generated with E. coli ribosomal RNA (supplied with the kit), and the range was 1 to 50 ng/ml.2 The standard and all samples were diluted in TE buffer and mixed with RiboGreen (Molecular Probes, Inc.) that had been diluted 2,000-fold. Fluorescence emission intensity was measured using disposable cuvettes, and the gain and range settings were adjusted to medium and 10,000, respectively. Relative fluorescence units (RFU) were recorded and plotted as a function of RNA added (Figure 1, R2=.988), and this linear relationship was used to determine the RNA concentration of samples.

When E. coli ribosomal RNA was treated with DNase-free RNase3 for 1 hour at 37 C, no RFU was detected. If samples were treated with DNase-free RNase, there was a 97% decrease in RFU. These results corroborated the idea that fluorescence emissions were being generated by RNA and not DNA.

RNA quantitation is a crucial feature of many molecular measurements. Prevotella bryantii endoglucanase mRNA was detected with slot-blots using a fluorochrome-labeled probe (Figure 2). Accurate quantitation of RNA was critical, and the VersaFluor fluorometer was used reliably in determining RNA concentrations.


References
1. Wen, Z. and Morrison, M., AEM, 62, 38263833 (1996).

2. Molecular Probes, Inc. Product Information Sheet, RiboGreen RNA quantitation reagent (1997).

3. Ausubel, F., et al., Short Protocols in Molecular Biology. A1-51 (1997).


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. Quantitation of Cabergoline at Extremely Low Plasma Concentrations with a Triple Quadrupole Mass Spectrometer
2. Bioanalytical Method Intraday Validation for the Quantitation of Paroxetine in Bovine Plasma using the TSQ Quantum Mass Spectrometer
3. Quantitation of the Protein MIA as a Marker for Chondrocytes in Research Samples
4. Comparison of Different Methods of mRNA Quantitation
5. Quantitation of DNA for Automated Sequencing Using the VersaFluor Fluorometer
6. Quantitation of Lymphangiogenesis Using the iCycler iQ Real-Time PCR Detection System and Scorpions Detection System, Rev A
7. Quantitation of GAD67 Gene Expression in Prefrontal Cortex of Schizophrenia Patients Using the iCycler iQ Detection System and Molecular Beacons, Rev A
8. Benchmark Plus Microplate Reader: Quantitation of Protein Concentration Using Two Different Colorimetric Assay Kits, Rev A
9. A Robust LC/MS/MS Method for the Quantitation of Very Long Chain Fatty Acids for Research into the Characterization of Peroxisomal Disorders
10. A new LC/MS/MS Research Method for Rapid Quantitation of Five Immunosuppressant Drugs, including Mycophenolic Acid (MPA)
11. Separation and Quantitation of R and S Enantiomers using Normal Phase Chromatography on an API 3000 LC/MS/MS System
Post Your Comments:
(Date:9/16/2014)... USA (PRWEB) September 16, 2014 “Nature ... create the features that we desire in optical design,” ... at Montana State University. “As we explore surfaces and ... of those lessons were presented in San Diego in ... Light: Light in Nature ” chaired by Shaw and ...
(Date:9/16/2014)... --The emerging field of molecular electronics could take ... enabling the construction of tiny circuits from molecular ... would take on the roles currently played by ... of researchers from five Japanese and Taiwanese universities ... small-scale electronics: a molecule called picene. In a ...
(Date:9/16/2014)... Cardioxyl Pharmaceuticals, Inc. announced today ... to be effective in animal models following oral (capsule) ... infusions that could pave the way for convenient outpatient ... at the Heart Failure Society of America,s (HFSA) 18 ... Vegas . The research, conducted in ...
(Date:9/16/2014)... Md. , Sept. 16, 2014   ... biotechnology company developing novel anti-infective biologic and drug ... and diseases, announced today the completion of the Company,s ... which highlighted its irritable bowel syndrome (IBS) ... constipation-predominant IBS (C-IBS). In the keynote ...
Breaking Biology Technology:Nature’s designs inspire research into new light-based technologies 2Nature’s designs inspire research into new light-based technologies 3Nature’s designs inspire research into new light-based technologies 4Nature’s designs inspire research into new light-based technologies 5The future face of molecular electronics 2Cardioxyl Presents Oral Heart Failure Treatment Data For Its Experimental HNO Prodrug At HFSA 18th Annual Scientific Meeting 2Synthetic Biologics' Statin-Class Candidate for Constipation-Predominant Irritable Bowel Syndrome Detailed at Company's Investor Day 2Synthetic Biologics' Statin-Class Candidate for Constipation-Predominant Irritable Bowel Syndrome Detailed at Company's Investor Day 3
... European Commission,s Joint Research Centre (JRC) has just ... a representative range of 25 different types of ... ensure consumer protection and confidence in many innovative ... most promising technological developments. By enabling a harmonised ...
... Feb. 14, 2011 New England Biolabs has expanded its ... One Taq Hot Start DNA Polymerases. Optimized blends of ... these new products offer robust amplification across a wide range ... exonuclease activity of Deep Vent increases the fidelity and robustness ...
... N.C., Feb. 14, 2011 India,s premier CRO, ... Society of India & Congress of Neurological Surgeons ...  The event was a convergence of leading neuroscientists, ... across the globe to discuss advances made in ...
Cached Biology Technology:Small material, big impact 2New England Biolabs Introduces OneTaq™ DNA Polymerase, a Robust Solution for Routine and Difficult PCR 2Max Neeman International Initiates Advisory Committee to Advance and Improve the Conduct of Complex Neurological Trials in India 2
(Date:9/16/2014)... Health at the Mailman School of Public Health are ... and prenatal exposure to two phthalates used in a ... the journal Environmental Health Perspectives . , ... levels of the chemicals, butylbenzyl phthalate (BBzP) and di-n-butyl ... increase in risk of developing asthma between age 5 ...
(Date:9/16/2014)... maternal morbidity amongst women in Australia is increased ... published today (17 September) in BJOG: An ... Australians generally enjoy high standards of living; however, ... in particular between indigenous and non indigenous Australians. ... independent impact of socioeconomic position on severe maternal ...
(Date:9/16/2014)... American Menopause Society (NAMS) has published its key, ... womenon everything from hot flashes to heart disease. ... Recommendations for Clinical Care of Midlife Women," was ... Menopause . This is the first, comprehensive set ... women freely available to all clinicians who care ...
Breaking Biology News(10 mins):Phthalates heighten risk for childhood asthma 2Phthalates heighten risk for childhood asthma 3New study examines the impact of socioeconomic position & maternal morbidity in Australia 2New study examines the impact of socioeconomic position & maternal morbidity in Australia 3
... Cambridge, Mass. - August 10, 2011 - Between conception ... meters long, looping and coiling within the tiny abdomen. ... loops into the same formationhowever, until now, it has ... experimental observations, biological and biophysical manipulations, theory, and computation, ...
... help overcome a major barrier to the use of gene ... reported development and successful initial laboratory tests of such a ... David Kaplan and colleagues note that gene therapy ... requires safe and efficient carriers or "vectors." Those carriers ...
... , Working out the structure of a complex formed ... be key in understanding how an antibiotic-producing organism controls ... example of how other antibiotic producers regulate export to ... certain microorganisms is a complex and highly regulated process, ...
Cached Biology News:Gut coils with help from its elastic neighbor 2Gut coils with help from its elastic neighbor 3Student brings home new expertise to answer question in antibiotic resistance 2Student brings home new expertise to answer question in antibiotic resistance 3
... esters have moderate chemiluminescence efficiencies and the ... They have the added benefit that they ... to the background signal. This benefit has ... sensitivity immunoassay applications. These compounds can also ...
... Kit for Kilo-Sequencing is designed to aid ... into the multiple cloning sites (MCS) of ... or phagemid vectors (pUC18/19). The kit is ... the target DNA, thereby progressively moving the ...
... client/server version of Freezerworks Unlimited includes 2 clients ... and facilitates the sharing of freezers by multiple ... Network Client/Server ... 13 in monitor with 256 colors; OS 8.6 ...
Immunogen: Synthetic peptide (KLH conjugated): L(506) S K S S S Q I R M S G L P A D N L A T(525) Storage: -20 C, Avoid Freeze/Thaw Cycles...
Biology Products: