Biology Reference
In-Depth Information
[s]
Ladder
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6 Sample 7
Sample 8
Sample 9 Sample 10 Sample 11 Sample 12
68
66
64
62
60
58
56
54
52
50
48
46
44
42
40
38
36
34
32
30
28
26
24
22
20
[FU]
45
40
35
30
25
20
15
10
5
0
[FU]
45
40
35
30
25
20
15
10
5
0
Sample 1
Sample 2
23S
23S
16S
16S
20
25
30
35
40
45
50
55
60
65
[s]
20
25
30
35
40
45
50
55
60
65 [s]
FIGURE 6.1
Gel-like image and example electropherograms of B. subtilis total RNA samples analyzed
using an RNA 6000 Nano LabChip Kit (Agilent Technologies) on the Agilent 2100
Bioanalyzer. The high degree of integrity of the RNA preparations is indicated by the rRNA
precursor bands visible above the 23S rRNA band. FU, fluorescence units.
Set (Qiagen). Subsequently, the samples are purified and the spin column-based
RNA Clean-Up and Concentration Kit from Norgen Biotek has been shown to retain
the small RNA species. RNA concentration and purity are then determined spectro-
photometrically, if possible by means of a NanoDrop instrument (Thermo Scien-
tific). RNA purity is a critical factor because contaminants can strongly impair
the efficiency of cDNA synthesis and labelling; therefore, the absorbance ratio of
A
260nm
/
A
280nm
should be
2 (low values indicate protein or phenol contamination)
and the absorbance ratio of
A
260nm
/
A
230nm
should be
>
1.8 (low values can indicate
various compounds such as phenol, guanidine thiocyanate or polysaccharides).
Finally, the integrity of the RNA preparations needs to be validated using the Agilent
Bioanalyzer (Agilent Technologies) or similar instrument, used according to the
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