Stanta, G.
Supplemental Table 1: Methods and Results of the used protocols for DNA extraction from
FFPE samples
Protocol, digestiona,b
Lab
DNAYield (g)
(A260/280 nm )
Protocol Type 1 [1, 4], 55°C 72 h, 1.0 µg/µl
Protocol Type 1, 55°C 2h or 37°C 16 h, 1.0 µg/µl
Protocol Type 1, 55°C 16h, 4.2 µg/µl
Protocol Type 1, 37°C 16h, 0.2 µg/µl
Protocol Type 1 [3], 55°C, 48 h, 1.0 µg/µl
Protocol Type 1, 56°C 16h, 1.8 µg/µl, followed
1
2
3
4
5
11
by Qiagen micro spin columns (protocol Type 3)
Protocol Type 2, 55°C 17h, 1.8 µg/µl
Protocol Type 2 [2], 56°C 16h, 3.3 µg/µl
Protocol Type 2, 37°C 48h, 0.95 µg/µl (only
6
7
8
colon sample) and Protocol type 3 QIAamp DNA
colon
ovary
lung 1
lung 2
87.0
25.6
50.8
26.0
(1.79)
(1.79)
(1.80)
(1.71)
80.5
37.4
92.5
52.3
(1.80)
(1.56)
(1.72)
(1.64)
72.8
16.4
27.4
15.6
(1.77)
(1.73)
(1.68)
(1.70)
----
33.0
33.0
13.0
----
(1.83)
(2.09)
(2.07)
51.4
18.5
26.7
25.7
(1.87)
(1.89)
(1.74)
(1.86)
49.1
8.1
15.6
16.9
(1.87)
(1.78 )
(1.88)
(1.89)
237.2
70.0
124.9
133.8
(1.41)
(1.09)
(1.20)
(1.20)
145.5
101.5
163.1
185.7
(1.10)
(0.83)
(1.05)
(1.08)
274.4
3.0
19.4
20.6
(1.35)
(1.73)
(1.88)
(1.85)
33.4
4.0
11.2
13.6
(1.99)
(1.75)
(1.97)
(1.91)
103.4
12.7
19.7
20.6
(2.00)
(2.01)
(2.03)
(2.03)
44.0
8.8
15.6
18.6
----
(1.77)
(1.74)
(1.74)
69.6
10.6
42.8
36.9
(1.43)
(1.29)
(1.42)
(1.42)
Mini Kit
Protocol type 3, Kit-Qiagen QIAamp DNA Mini
9
Kit3
Protocol type 3, Kit-Qiagen QIAamp DNA Mini
10
Kit
Protocol Type 3, Kit-Qiagen DNeasy tissue3
Protocol Type 3, Kit-Macherey Nagel
NucleoSpin Tissuec
12
13
1
Stanta, G.
a
Protocols: type 1, DNA extraction with precipitation of the DNA; type 2, DNA extraction without
further precipitation or purification; type 3, DNA extraction using silica based absorption columns.
Citations in brackets.
b
Time, temperature and final concentration of Proteinase K in the digestion step.
c
Proteinase K treatment according to manufactures instructions
For participants 2, 3, 4, 6 and 8 there are no references reporting the protocols used for DNA
extractions. Later on are reported the digestion buffer composition for the previously reported
participants.
2
Stanta, G.
Supplemental Table 2: protocol type, digestion buffer composition for DNA and RNA
extraction from FFPE samples for protocols not previously published.
DNA
Protocola, digestionb
Protocol Type 1, 55°C 2h or 37°C 16 h, 1.0 µg/µl
Participant
2
Digestion buffer: 1X PCR buffer II (without MgCl2) Applied Biosystems (Cat N°
F06312 for 10X stock solution)
Protocol Type 1, 55°C 16h, 4.2 µg/µl
3
Digestion buffer: 10 mM Tris-HCl pH 8.3, 1 mM EDTA, 0.2% Tween 20.
Protocol Type 1, 56°C 16h, 1.8 µg/µl, followed by Qiagen micro spin columns
11
Digestion Buffer: 10 mM Tris-HCl pH 8.5, 100 mM NaCl, 1 mM EDTA, 0.5%
Tween 20, 0.5% NP-40, 20 mM DTT. Enzyme inactivation: 5 min @95°C
Protocol Type 1, 37°C 16h, 0.2 µg/µl
4
Digestion buffer 100 mM EDTA, 2.5% sarkosyl (Na salt of N-lauroylsarcosine.
Protocol Type 2, 55°C 17h, 1.8 µg/µl
6
Digestion buffer: 10 mM Tris-HCl pH 8.3, 1 mM EDTA, 0.5% Tween 20.
Protocol Type 2, 37°C 48h, 0.95 µg/µl (only colon sample)
8
Digestion buffer: 1X PCR buffer II (without MgCl2) Applied Biosystems (Cat N°
F06312 for 10X stock solution). Enzyme inactivation: 10 min @94°C.
RNA
Protocola, digestionb
Protocol Type 4, ON 56°C, 0.42 µg/µl
Participant
4
Buffer composition: 10 Mm Tris-HCl pH8, 1 mM EDTA, 2% SDS.
Protocol Type 5, digestion homemade ON 55°C, 0.95 µg/µl
8
Buffer composition: 20 mM Tris-HCl pH8, 20 mM EDTA, 2% SDS
a
Protocols: type 1, DNA extraction with precipitation of the DNA; type 2, DNA extraction without
further precipitation or purification; type 4, homemade protocol for RNA extraction followed by
precipitation of the RNA; type 5, RNA extraction with monophasic commercial solutions and
isopropanol precipitation
b
Temperature, time, and final concentration of Proteinase K in the digestion step
3
Stanta, G.
Supplemental Table 3: Methods and Results of the used protocols for RNA extraction from
FFPE samples
Protocol, digestiona,b
Lab
DNA Yield (g)
(A260/280 nm )
Protocol Type 4 [5, 6], 55°C 45h, 6 µg/µl
Protocol Type 4 [5, 6], 55°C ON, 6 µg/µl
Protocol Type 5, digestion homemade and Trizol
2
5
8
ON, 0.95 µg/µl
Protocol Type 5, Prot K digestion in lysis buffer
11
(Biozym) and RNA-BEE (Bioconnect), 56°C ON,
colon
ovary
lung 1
lung 2
16.7
1.5
8.9
9.2
(2.0)
(1.73)
(1.92)
(1.89)
22.2
1.8
6.1
22.2
(1.86)
(1.80)
(1.94)
(1.99)
46.9
1.6
23.0
44.6
(1.85)
(1.62)
(1.84)
(1.89)
30.0
3.6
18.7
37.5
(1.99)
(1.75)
(1.85)
(1.78)
10.3
0.2
3.1
8.2
(1.7)
(1.15)
(1.58)
(1.61)
11.7
4.9
9.3
4.7
(2.0)
(1.85)
(1.79)
(1.87)
61.0
6.6
19.8
24.7
(1.93)
(1.78)
(1.90)
(1.96)
48.4
2.5
39.9
46.4
(1.90)
(1.79)
(1.99)
(1.98)
33.4
4.5
7.3
7.2
(2.0)
(1.79)
(1.85)
(1.87)
60
9.2
40.0
40.0
(--)
(1.89)
(1.97)
(1.99)
1.5 µg/µl
Protocol Type 6, Kit-Gentra Purescript RNA 65°C
3
2h
Protocol Type 6, Kit-Qiagen RNeasy FFPE and
4c
Protocol Type 4 ON 56°C, 0.42 µg/µl
Protocol Type 6, Kit Roche High Pure RNA 55°C
6
17h
Protocol Type 6, Kit-Qiagen RNeasy FFPE 55°C
7
15’
Protocol Type 6, Kit-Roche High Pure RNA 55°C
9
ON
Protocol Type 6, Kit-Qiagen RNeasy FFPE ON
55°C
a
12
Protocols: type 4, RNA extraction with phenol extraction and isopropanol precipitation-
homemade protocols; type 5, RNA extraction with mono-phase commercial solutions and
isopropanol precipitation; type 6, RNA extraction using silica based columns for purification.
Citations in brackets.
b
Time, temperature and final concentration of Proteinase K in the digestion step. ON, over night
c
This participant used protocol type 6 only for colon cancer sample, as underlined.
4
Stanta, G.
Supplemental Table 4: Dependency of RNA yield on tumor tissue and isolation method.
RNA
yield
> 50 µg
colon cancer
>= 40 µg
Participant 8 (homemade Trizol), participant 7 (Kit-Qiagen RNeasy)
>= 30 µg
Participant 11 (commercial RNAzol), participant 9 (Kit-Roche High Pure RNA)
< 30 µg
Participant 5 (homemade), participant 2 (homemade), participant 4 (Kit-Qiagen
Participant 12 (Kit-Qiagen RNeasy), participant 6 (Kit Roche High Pure)
RNeasy FFPE), participant 3 (Kit-Gentra Purescript RNA)
RNA
lung cancer
yield
>= 40 µg
both samples: Participant 12 (Kit-Qiagen RNeasy), participant 7 (Kit-Qiagen RNeasy)
>= 40 µg
one sample: Participant 8 (homemade Trizol)
>= 20 µg
Participant 11 (commercial RNAzol), participant 5 (homemade protocol), participant 6
(Kit Roche High Pure)
< 10 µg
Participant 2 (homemade Trieste protocol), participant 4 (homemade), participant 9
(Kit-Roche High Pure RNA), participant 3 (Kit-Gentra Purescript RNA)
RNA
ovarian cancer
yield
> 9 µg
Participant 12 (Kit-Qiagen RNeasy)
> 6 µg
Participant 6 (Kit Roche High Pure)
> 4 µg
Participant 4 (home made), participant 9 (Kit-Roche High Pure RNA)
< 4 µg
Participant 11 (commercial RNAzol), participant 7 (Kit-Qiagen RNeasy), participant 5
(homemade), participant 8 (homemade, Trizol), participant 2 (homemade), participant
3 (Kit-Gentra Purescript RNA)
5
Stanta, G.
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Higuchi R (1989) Simple and rapid preparation of samples for PCR. In: PCR technology,
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Pauluzzi P, Bonin S, Gonzalez Inchaurraga MA, Stanta G, Trevisan G (2004) Detection of
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Stanta G, Bonin S, Perin R (1998) RNA extraction from formalin-fixed and paraffin-
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Stanta G, Schneider C (1991) RNA extracted from paraffin-embedded human tissues is
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6