Like that of DNA, electrophoresis of RNA is usually carried out in agarose gels, and the principle of the separation, based on size, is the same. Often, one requires a rapid method for checking the integrity of RNA immediately following extraction and before deciding whether to process it further. This can be achieved easily by electrophoresis in a 2% agarose gel in about 1 h. Ribosomal RNAs (18 S and 28 S) are clearly resolved and any degradation (seen as a smear) or DNA contamination is seen easily. This can be achieved on a 2.5–5% acrylamide gradient gel with an overnight run. Both these methods involve running native RNA. There will almost certainly be some secondary structure within the RNA molecule owing to intramolecular hydro gen bonding (see, for example, the clover leaf structure of tRNA, Figure1). For this reason, native RNA run on gels can be stained and visualised with ethidium bromide. However, if the study objective is to determine RNA size by gel electrophoresis, then full denaturation of the RNA is needed to prevent hydrogen bond formation within or even between polynucleotides that will otherwise affect the electrophoretic mobility. There are three denaturing agents (formaldehyde, glyoxal and methylmercuric hydroxide) that are compatible with both RNA and agarose. Either one of these may be incorporated into the agarose gel and electrophoresis buffer, and the sample is heat denatured in the presence of the denaturant prior to electrophoresis. After heat denaturation, each of these agents forms adducts with the amino groups of guanine and uracil, thereby preventing hydrogen bond reformation at room temperature during electrophoresis. It is also necessary to run denaturing gels if the RNA is to be blotted (Northern blots) and probed, to ensure that the base sequence is available to the probe. Denatured RNA stains only very weakly with ethidium bromide, so acridine orange is commonly used to visualise RNA on denaturing gels. However, it should be noted that many workers will be using radiolabelled RNA and accordingly identify bands by autoradiography. An example of the electrophoresis of RNA is shown in Figure2.

Fig1. Clover-leaf secondary structure of yeast tRNA Phe . A single strand of 76 ribonucleotides forms four double-stranded ‘ stem’ regions by base-pairing between complementary sequences. The anticodon will base-pair with UUU or UUC (both are codons for phenylalanine); phenylalanine is attached to the 3′ end by a specific aminoacyl tRNA synthetase. Several ‘unusual’ bases are present: D, dihydrouridine; T, ribothymidine; ψ, pseudouridine; Y, very highly modified, unlike any ‘normal’ base. mX indicates methylation of base X (m 2 X shows dimethylation); Xm indicates methylation of ribose on the 2′ position; p, phosphate.

Fig2. Separation of yeast RNA on a 1.5% agarose gel. (Courtesy of Dr Tomas Masek, Department of Genetics and Microbiology, Charles University, Prague, Czech Republic.)

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المزيد

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علماء يكتشفون انخفاض صلابة الألماس عند تحويله إلى مقياس نانوي

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المزيد

مواضيع ذات صلة

Microchip Electrophoresis

Pulsed-Field Gel Electrophoresis

3′ Cleavage and Polyadenylation of Pre-mRNAs Are Tightly Coupled

Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs

SR Proteins Contribute to Exon Definition in Long Pre-mRNAs

DNA Sequencing Gels

Nuclear Bodies Are Functionally Specialized Nuclear Domains

Chain Elongation by RNA Polymerase II Is Coupled to the Presence of RNA-Processing Factors

Spliceosomes, Assembled from snRNPs and a Pre-mRNA, Carry Out Splicing

During Splicing, snRNAs Base-Pair with Pre-mRNA

Transport of mRNA Across the Nuclear Envelope

Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions