Quality assessment of total RNA








RNA quality control using the Agilent's 2100 Bioanalyzer

     the Ugly
     (RINdb)
     Bioanalyzer analysis.
     not the same, why not?
     microarray experiments


Quality assessment of total RNA sample

Total RNA run on a denaturing agarose gel or on Agilent's 2100 Bioanalyzer shows
two distinct ribosomal peaks corresponding to either18S and 28S for eukaryotic RNA
or 16S and 23S for prokaryotic RNA and a relatively flat baseline between the 5S and
18S ribosomal peaks.

The major human rRNA species (18S and 28S) are synthesized by cleavage from a
common 13 kb transcription unit which is part of a 40 kb tandemly repeated genomic
unit. The human genome contains many hundreds of these repeated rRNA genes
(335 rRNA genes, according to NCBI Assembly 36), which when transcribed result in
extremely high levels of rRNA transcripts. There can be as many as 10 million copies
of rRNA per cell. In addition to the many hundreds of rRNA genes, the extremely high
stability of the rRNA transcripts (4-5 days) strongly contributes to their high levels
within the cell.




































The fact that the 18S and 28S rRNA species are derived from the same precursor
molecule means that there are exactly the same number of copies of both molecules
in the cell. Traditionally, the intensity of these rRNA bands on denaturing agarose
gels have been used to calculate a ratio that served as an indication of RNA integrity.
A 28S/18S ratio of two is considered to be good quality RNA.



















If one would consider the amount of bases of these major rRNA species (for human,
the 28S rRNA species has 5034 bases and the 18S rRNA species has 1870 bases)
the exact ratio should be 2.7 and for mouse rRNA  (see table) this should be 2.5. The
reason that the 28S rRNA band or peak is seen as more intense despite the fact that
there are exactly as many molecules as for the 18S rRNA is because the detection of
rRNA depends on the binding (intercalation) of dye to the RNA and this depends on
the number of bases present in each molecule. So twice as many bases will bind
twice as many dye molecules, which will result in a twice as intense signal intensity
of the band on gel or the electropherogram peak of the Bioanalyzer























The major problem with traditional denaturing gel electrophoresis is the large
amount of RNA required to detect a clearly visible band. Depending on the system
used, this could be a few hundred nanograms up to even a microgram of total RNA
that would be required to visualize the rRNA bands. It is clear that if small needle
biopsies or laser capture material is to be used, these amounts of RNA could not be
used up just for checking the quality of the RNA. In this sense, the Bioanalyzer is a
great asset to the modern diagnostic lab for those samples of which only limited
amounts of sample for extracted RNA are available to begin with.






Next: Agilent's 2100 Bioanalyzer, Lab on a Chip for the quality control of RNA
RNA QUALITY CONTROL
The invaluable help of Bieke Vanherle, Erika Timmer and Frank Beijers
is acknowledges in performing the experiments
described in this section.
Biosynthesis of rRNA transcripts. Small arrows indicated by letters A-D signify positions of
endonuclease cleavage of RNA precursors. Cleavage of the 41S precursor at B generates two
products: 20S + 32S. Following cleavage of the 32S precursor at D, and excision of the small
5.8S rRNA, hydrogen bonding takes place between the 5.8S rRNA and a complementary central
segment of the 28S rRNA. The approximately 6 kb of RNA sequence originating from the external
and internal transcribed spacer units (ETS, ITS1 and ITS2) are degraded in the nucleus. S is the
sedimentation coefficient, a measure of size. The figure was adapted from: Human Molecular
Genetics 2 from Tom Strachan and Andrew P. Read.
Traditional separation of total RNA on denaturing agarose gel
electrophresis followed by ethidium bromide staining. The 28S/18S
rRNA bands in this figure have an intensity ratio around 2 that
would be considered good to very good quality RNA.
Species
rRNA
Size (bases)
Ratio 28S/18S
       
Human
28S
5034
2.69
  18S
1870
 
       
Mouse
28S
4729
2.53
  18S
1869
 

Table: Size of human and mouse rRNA molecules and the theoretical ratios of
their 28S and 18S rRNAs.
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