Report on “Random dilution” experiment with Agilent ribosomal microarray
Experiment performed Aug 5 2003.
Version 1, Aug 13, 2003
Experimental setup
Outline of the experiment:
How far can we dilute PCR fragments and still be able to detect them in a mix of PCR fragments of
different concentrations?
PCR fragments
About 250 bacterial ATCC strains were amplified with 16S ribosomal broad range primers 8F x
1391R. PCR products were purified using a 96-wells format QIAquick purification kit. Concentration
was determined with the PicoGreen Assay kit in a fluorescent microplate reader. The 192 PCR
products with the highest concentration were selected, and all concentrations were brought to 2 ng/ul.
Common reference pool
From each of the 192 samples, 10 ul was taken and pooled to create a common reference pool. The
concentration was 192 x 10 x 2 = 3830 ng in 1920 ul. The pool was concentrated by QIAquick column
purification to obtain a final concentration of 96 ng/ul. So per species the DNA concentration is 0.5
ng/ul. This pool was called CR3 (version 3; does not include B. subtilis/E.coli/yeast/human DNA).
Dilution and randomization
Serial dilutions were made of all 192 selected PCR products in microplates, to obtain 6 dilutions (see
table). To facilitate the randomization, dilutions were performed into colored microtubes. The dilutions
were randomized per strain and subsequently pooled. From each dilution 25 ul was taken to obtain 6
pools, which were labeled A-F.
Dilution
1
2
3
4
5
6
undiluted
10x diluted
100x diluted
1000x diluted
10,000x diluted
water
Concentration
(ng/ul)
2
0.2
0.02
0.002
0.0002
0
Color
Blue
Purple
Red
Orange
Yellow
Clear
CP wrote a randomization program which ensured that each of the 6 final pools consisted of 32
samples per dilution, so that the final DNA concentration per pool was the same.
Each pool consists of 192 species-specific PCR products. The final DNA concentration was 1777.7 ng
in 4800 ul.
25 ul x 32 species x 2 ng/ul =
25 ul x 32 species x 0.2 ng/ul =
25 ul x 32 species x 0.02 ng/ul =
25 ul x 32 species x 0.002 ng/ul =
25 ul x 32 species x 0.0002 ng/ul =
25 ul x 32 species x 0 ng/ul =
Total per pool
1600 ng
160 ng
16 ng
1.6 ng
0.16 ng
0 ng
1777.7 ng in 4800 ul
The 6 pools were concentrated by speedvac evaporation and QIAquick column purification to a final
concentration of 30-40 ng/ul as determined by spec and gel.
12 strains
12 strains
6 dilutions
A
B
C
D
E
F
Before randomization; Plate 1; Strains 1-96
After randomization; Plate 1; Strains 1-96
Labeling and hybridization
The CR3 pool was supplemented with an equimolar amount of S. cerevisiae ribosomal DNA (1000 bp
fragment of pEB26), and was labeled with Cy3. The 6 random dilution pools were labeled with Cy5.
Hybridization was performed according to the following scheme (final volume 300 ul).
Hybe
Slide number *)
Water
A
16011874010004 B
85.3 ul
B
16011874010004 N
85.6 ul
C
16011874010005 B
84.1 ul
D
16011874010005 N
76.6 ul
E
16011874010007 B
81.0 ul
F
16011874010007 N
82.8 ul
*) B = barcode side; N = non-barcode side
CR3 (Cy3)
Random pool
4.7 ul of 64 ng/ul
4.7 ul of 64 ng/ul
4.7 ul of 64 ng/ul
4.7 ul of 64 ng/ul
4.7 ul of 64 ng/ul
4.7 ul of 64 ng/ul
10.0 ul of 30.0 ng/ul
9.7 ul of 31.3 ng/ul
11.2 ul of 26.7 ng/ul
18.8 ul of 16.0 ng/ul
14.3 ul of 21.0 ng/ul
12.5 ul of 24.0 ng/ul
Buffer and
Standards
200 ul
200 ul
200 ul
200 ul
200 ul
200 ul
In all six hybes the final concentration of the CR3 pool was 1 ug/ml, which was 5.2 ng/ml per species.
The DNA concentration for each of the Random Dilution pools was also 1 ug/ml. For any undiluted
species, there would be 28 ng/ml present, for the 1:10 diluted species this would be 2.8 ng/ul, etc.
Hybridization was performed at 60°C for 17 h. Slides were washed 10 min in low stringency buffer
(with 6x SSC) at room temp and 10 min in high stringency buffer (with 0.6x SSC) at 4°C.
Slides were scanned at PMT 5% (both red and green channels).
Results
Average of all 10,807 probes
Average
gBSubSignal
rBSubSignal
Ratio R/G
A
2576.9
11736.7
3.93
B
2186.5
11774.5
4.70
C
1917.8
12536
5.54
D
1486.5
13046.4
7.81
E
924.6
12928.1
11.26
F
1312.5
11895.7
7.93
C
3242.0
36651.5
11.44
D
2084.4
36745.4
17.83
E
1597.4
39207.0
25.32
F
2530.1
37438.1
15.09
Average of all “universal” probes (n=259)
Average
gBSubSignal
rBSubSignal
Ratio R/G
A
5153.7
34261.5
6.68
B
4228.8
36849.7
8.84
The average red signals were 4-20 times higher than the green signals. How to explain this, when the
input concentrations are the same for each channel?
Ranking (see attached table)
Top 50 ranked on gBGSubSignal in hybe A: many S. cerevisiae probes (yeast was present in the
green but not in the red label).
Top 50 ranked on rBGSubSignal in hybe A: B. subtilis / E. coli tiled probes and universal probes
Top 50 ranked on ratio R/G in hybe A: lot of garbage caused by probes with very low signals; after
filtering for this, the top 50 consists of E. coli / B. subtilis tiled sequences and node probes.
Hybridization to species-specific probes
To analyze the results, probes were selected forfilling the following two criteria:
1. species-specific probes obtained with the BEST algorithm (most species are represented on the
array with 1-5 probes)
2. species that were represented in the CR3 pool (containing 192 species)
Of the 10,000 probes, 497 probes forfilled these criteria.
For each probe, the R/G ratio’s of the BsubSignals were put back into order, so that the hybes with
the highest concentration of the corresponding PCR product (undiluted; dilution 1) were all in one
column, the 10x dilutions were in a second column, etc. Then a XY scatter plot was made, giving the
ratio’s of all the 497 probes per dilution.
Average R/G BBSubSignal ratio per dilution:
Dilution#
Dilution
Average
Standard
deviation
1
Undiluted
7.52
4.53
2
10x
4.82
2.20
3
100x
4.75
4.13
4
1000x
3.83
2.65
5
10000x
5.20
4.29
6
water
4.30
3.85
R/G ratio's Species BEST probes
20.00
R/G ratio BGSubSignals
(1) Undiluted
15.00
(2) 10x diluted
(3) 100 x diluted
(4) 1000x diluted
10.00
(5) 10,000x diluted
(6) water
Averages
AVE+StandDev
5.00
AVE-StandDev
0.00
0
1
2
3
4
5
6
7
Dilution
E.coli tiled probes
100000
10
9
10000
8
6
5
100
4
3
10
2
1
1
0
Position on 16S gene
R/G ratio
1000
0
10
5
21
0
31
5
42
0
52
5
63
0
73
5
84
0
94
10 5
5
11 0
55
12
6
13 0
65
BGSubSignal
7
gBGSubSignal pool A
rBGSubSignal pool A
Ratio R/G
B. subtilis tiled probes
100000
12
10
10000
BSubSignal
8
1000
6
100
gBSubSignal Hybe A
rBGubSignal Hybe A
Ratio R/G Hybe A
4
10
2
0
5
95
18
5
27
5
36
5
45
5
54
5
63
5
72
5
81
5
90
5
99
10 5
8
11 5
7
12 5
6
13 5
5
14 5
45
1
Position
10000.00
1.00
1000.00
0.50
100.00
0.00
10.00
-0.50
1.00
-1.00
0.10
-1.50
Position
10LogRatio R/G
1.50
12
0
24
0
36
0
48
0
60
0
72
0
84
0
96
10 0
8
12 0
0
13 0
2
14 0
4
15 0
6
16 0
80
100000.00
0
BGSubSignal
S. cerevisiae tiled sequences
gBGSubSignal Pool A
rBGSubSignal Pool A
10logRatio R/G
Escherichia coli tiled sequences
No E. coli 16S DNA was included in the common reference pool, nor in the random dilution pools,
because the concentration of the purified PCR fragment was too low.
The hybridization signals of these probe was therefore expected to be low, except for conserved
regions, because they could bind to sequences from other species. Also, there were some other
Gamma Proteobacteria included in both types of pools, so cross hybridization can be expected.
The plot of the hybridization signals against the position of the E. coli 16S gene shows low green
signals and much higher red signals. This could be caused by the fact that the red labeled dilution
pools contain some fragments that are present in 5x higher concentration than in the green labeled
common reference pool.
Bacillus subtilis tiled sequences
The Bacillus plot is similar to the E.coli one. The graph shows dips in the signals around position 450460, at 980-1000 and 1260-1280. This could be caused by accessibility problems (these probes do
not have a tether).
Saccharomyces cerevisiae tiled sequences
The CR3 used in this experiment had extra yeast 18S DNA added. The fragment added was the
amplification product of the M13 primers on the insert of plasmid pEB26, containing F1 x F3 (position
555-1550 of GenBank Accession number J01353, on which the tiled sequence is based).
As expected, the hybridization signals outside of this region are low. The green signals become high
around position 535 (the first oligo to contain the F1 primer completely) and drop around 1530 (this
oligo still contains the complete F3 region, but might not be accessible because these arrays are
printed without tether), as expected.
In the random diluted pools (labeled with Cy5, so red channel), no yeast DNA was added.
Surprisingly, the red signals from the hybe with Pool A follow the exact same pattern as the green
signals. The red signals do not reach the same hybridization strength as the green signals, as shown
by the 10logRatio R/G which is <1 in this part of the molecule, but the signals are high at the same
position as the green signals.
Could this mean that red-labeled fragments bind to green-labeled fragments during the hybe????
Can we eliminate this effect by labeling only one strand of the PCR fragment (e.g.unifirectional
amplification with primer 1391?
There is a remarkable drop in both the red and the green signal at position 1025, which is only found
in the hybe with Pool A. All other 5 hybes do not show this dip.
CHECK THE PICT OF THE ARRAY TO SEE IF THERE WAS A PROBLEM
There is also an interesting increase in the signal at positions 1585-1630, so outside the labeled
region. The sequence of this region is:
GCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTAGTA
CCGATTGAATGGCTTAGTGAG
The high hybridization signal is probably explained by the fact that this region contains a highly
conserved sequence, the underlined sequence is primer 1391 (universal primer), so also bacterial
labeled products can hybridize to this region.
Main conclusions


The results from this experment do not make much sense. There is not a clear relationship
between amount of labeled product and strength of hybridization signal.
Maybe the confusing results are caused by hybridization of not only the labeled fragment to its
corresponding probe on the array, but also of hybridization of fragments to each other. In a next
experiment we could study the effect of bi- and unidirectional labeling, maybe by using shorter
and longer differently labeled BS or EC fragments.