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:7/10/2014)... (PRWEB) July 10, 2014 Terascala, the ... announced that Alan Swahn, a former vice president of ... team as vice president of marketing. Terascala’s software when ... and NetApp create the highest performance and most reliable ... appointed to guide Terascala’s channel expansion and broaden its ...
(Date:7/10/2014)... 10, 2014 Senior supply chain management ... associations, addressed the challenges of “Reducing Cost, Lead Time, ... through strategic sourcing. Describing the partnership of the Bio ... Chain Management Institute ( SCMI) of the University ... 24 at the Kroc Institute of Peace and Justice ...
(Date:7/10/2014)... , July 10, 2014 Research and ... of the "International Photonic Integrated Circuit (Monolithic ... to 2019" report to their offering. ... The concept of photonic integration traces its roots ... The promise of photonic integration went unexplored and ...
(Date:7/10/2014)... today in the journal PNAS (Proceedings of the ... of day and sleep deprivation have a significant effect ... looking at the best time of day to test ... for administering medicines effectively. , Researchers from the University ... investigated the links between sleep deprivation, body clock disruption ...
Breaking Biology Technology:Terascala Expands Executive Team 2Terascala Expands Executive Team 3Biotech & Biopharm Supply Chain Management Executives Launch BSMA in San Diego 2Biotech & Biopharm Supply Chain Management Executives Launch BSMA in San Diego 3Biotech & Biopharm Supply Chain Management Executives Launch BSMA in San Diego 4International Photonic Integrated Circuit (Monolithic Integration, Hybrid Integration, Module Integration) Market - Forecasts to 2019 2Time of day crucial to accurately test for diseases, new research finds 2
... thick material graphene maintains its high thermal conductivity when ... the material from a laboratory phenomenon to a useful ... in the April 9 issue of the journal ... physicists from the University of Texas at Austin, Boston ...
... ... and medical device companies. , ... Chicago, IL (PRWEB) April 8, 2010 -- ClearTrial ( www.cleartrial.com ), the ... demonstrating the software,s capabilities to drive greater efficiency in clinical operations and outsourcing to ...
... ... ISA Certified Arborists , ... (PRWEB) April 7, 2010 -- Michael Sitaras showcased his professional tree ... of the International Society of Arboriculture (ISA). , , ,Michael developed his ...
Cached Biology Technology:With support, graphene still a superior thermal conductor 2ClearTrial to Demonstrate How a Top 20 Sponsor Achieved -1 Percent Cost Variance Across Their $130M Clinical Portfolio at Major Industry Conference 2ClearTrial to Demonstrate How a Top 20 Sponsor Achieved -1 Percent Cost Variance Across Their $130M Clinical Portfolio at Major Industry Conference 3Michael Sitaras of SavATree Earns ISA Certified Arborist Designation 2
(Date:7/10/2014)... , July 1, 2014 Smart ... way for convenience and improved security: NXT-ID, Inc. (OTCBB: NXTD), ... GOOG ), Apple Inc. (NASDAQ: AAPL ... Corporation (NYSE: MA) NXT-ID, Inc., (OTCQB: NXTD) a biometric ... to announce that the second series of 30 second spots ...
(Date:7/10/2014)... July 3, 2014 According to ... Type (Swipe And Area), Material (Optical Prism, Pizeoelectric, Capacitive, ... Security and Banking & Finance), and Geography - Global ... MarketsandMarkets, the global Fingerprint Sensors Market is projected to ... CAGR of 16.8% from 2014 to 2020. ...
(Date:7/10/2014)... 2014 Fingerprint Cards, (FPC,s) ... win (DW). An Asian OEM has selected FPC1021 for a ... in August 2014.   FPC is proud to ... a planned date for start of mass production in August ... SEK 5M, for delivery in the first half of July. ...
Breaking Biology News(10 mins):Advantages for Digital Wallets Eliminate Need to Carry Credit Cards - Large Scale CNBC Ad Campaign for Latest Smart Wallet to Commence July 7th 2Advantages for Digital Wallets Eliminate Need to Carry Credit Cards - Large Scale CNBC Ad Campaign for Latest Smart Wallet to Commence July 7th 3Advantages for Digital Wallets Eliminate Need to Carry Credit Cards - Large Scale CNBC Ad Campaign for Latest Smart Wallet to Commence July 7th 4Advantages for Digital Wallets Eliminate Need to Carry Credit Cards - Large Scale CNBC Ad Campaign for Latest Smart Wallet to Commence July 7th 5Fingerprint Sensors Market worth $14.35 Billion by 2020 2Fingerprint Sensors Market worth $14.35 Billion by 2020 3FPC Wins DW and Initial Ramp Order for FPC1021 Touch Sensor for Flagship Smartphone from Asian OEM 2
... Energy in, energy out, it,s the basic equation to weight ... of Americans classified as overweight or obese1, a study in ... Education and Behavior examines how motivation might be a ... at the University of Kentucky and University of North Carolina ...
... BIO-key International, Inc. (OTC Bulletin Board: BKYI), a leader in finger-based biometric ... successful deployment of an FBI-compliant advanced authentication solution for law enforcement mobile ... , , ... , , ...
... use of antibiotics to treat bacterial infections causes a ... to the emergence and spread of resistant strains, forcing ... resistance. But what if it doesn,t have to ... American Society for Biochemistry and Molecular Biology,s annual meeting, ...
Cached Biology News:What's motivation got to do with weight loss? 2BIO-key(R) Delivers FBI-Compliant 2 Factor Authentication Solution for First Responders 2BIO-key(R) Delivers FBI-Compliant 2 Factor Authentication Solution for First Responders 3BIO-key(R) Delivers FBI-Compliant 2 Factor Authentication Solution for First Responders 4Putting bacterial antibiotic resistance into reverse 2
Recombinant Human HGF...
Phosphorylcholine hapten is conjugated to BSA protein by use of p-diazonium phenylphosphorylcholine....
Request Info...
6-(2,4-dinitrophenyl)aminohexanoic acid, succinimidyl ester (DNP-X, SE)...
Biology Products: