High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning.
To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods.
Masters
D B
DB
Children's Hospital, Boston, MA 02115.
Griggs
C T
CT
Berde
C B
CB
D000199:Actins / Q000235:genetics; D000703:Analog-Digital Conversion; D000818:Animals; D001345:Autoradiography / Q000379:methods*; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods*; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012339:RNA, Ribosomal, 28S / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D012044:Regression Analysis
D000199:Actins; D015345:Oligonucleotide Probes; D012339:RNA, Ribosomal, 28S