Evaluation of Reaction Products (E2065)
Quantification by UV Light AbsorbanceRNA concentration can be determined by measuring the ultraviolet light absorbance at 260 nm, however, any unincorporated nucleotides and template DNA in the mixture will affect the reading. Free nucleotides from the transcription reaction must be removed before the RNA concentration can be quantified. RNA solution can be read directly on a Nanodrop™ Spectrophotometer. The Nanodrop Spectrophotometer can directly read RNA concentrations from 10 ng/μl to 3000 ng/μl. For single-stranded RNA, 1 A260 is equivalent to an RNA concentration of 40 μg/ml. The RNA concentration can be calculated as follows:
A260 x dilution factor x 40 = __ μg/ml RNA
Analysis of Transcription Products by Gel ElectrophoresisTo evaluate transcript length, integrity and quantity, an aliquot of the transcription reaction should be run on an appropriate denaturing agarose or polyacrylamide gel. Transcripts larger than 0.3 kb can be run on agarose gels, whereas denaturing polyacrylamide gels (5–15%) are necessary for smaller transcripts. The gels should be run under denaturing conditions to minimize formation of secondary structures by the transcript.
- Preparation of denaturing gels
a. Denaturing agarose gel:
To make a 100 ml 1% denaturing agarose gel, add 1 gram agarose powder to 72 ml nuclease-free water. Melt the agarose and add 10 ml 10X MOPS buffer. Then, in a fume hood, add 18 ml fresh formaldehyde (37%), mix well. Pour the gel.
10X MOPS gel running buffer: 0.4 M MOPS (pH 7.0), 0.1 M sodium acetate, 10 mM EDTA
b. Denaturing PAGE/Urea Gel:
5–15% PAGE/Urea gel. We recommend using commercially available premade gels. Use standard TBE gel running buffer.
10X TBE buffer: 0.9 M T ris Base, 0.9 M Boric Acid, 20 mM EDTA.
- Gel electrophoresis
a. Mix 0.2–1 μg RNA sample with 5-10 μl of RNA Loading Dye (2X, NEB #B0363 ).
b. Denature the RNA sample and an aliquot of RNA marker by heating at 65–70°C for 5–10 minutes.
c. Pulse-spin prior to loading onto gel.
d. Visualizing RNA by staining the gel with SYBR® Gold or ethidium bromide.