Stanta, G.

Supplemental Table 1: Methods and Results of the used protocols for DNA extraction from FFPE samples

Protocol, digestiona,b / Lab / DNAYield (mg)
(A260/280 nm )
colon / ovary / lung 1 / lung 2
Protocol Type 1 [1, 4], 55°C 72 h, 1.0 µg/µl / 1 / 87.0 (1.79) / 25.6 (1.79) / 50.8
(1.80) / 26.0
(1.71)
Protocol Type 1, 55°C 2h or 37°C 16 h, 1.0 µg/µl / 2 / 80.5 (1.80) / 37.4 (1.56) / 92.5
(1.72) / 52.3
(1.64)
Protocol Type 1, 55°C 16h, 4.2 µg/µl / 3 / 72.8
(1.77) / 16.4 (1.73) / 27.4
(1.68) / 15.6
(1.70)
Protocol Type 1, 37°C 16h, 0.2 µg/µl / 4 / ----
---- / 33.0 (1.83) / 33.0
(2.09) / 13.0
(2.07)
Protocol Type 1 [3], 55°C, 48 h, 1.0 µg/µl / 5 / 51.4
(1.87) / 18.5 (1.89) / 26.7
(1.74) / 25.7
(1.86)
Protocol Type 1, 56°C 16h, 1.8 µg/µl, followed by Qiagen micro spin columns (protocol Type 3) / 11 / 49.1
(1.87) / 8.1 (1.78 ) / 15.6
(1.88) / 16.9
(1.89)
Protocol Type 2, 55°C 17h, 1.8 µg/µl / 6 / 237.2
(1.41) / 70.0 (1.09) / 124.9
(1.20) / 133.8
(1.20)
Protocol Type 2 [2], 56°C 16h, 3.3 µg/µl / 7 / 145.5
(1.10) / 101.5 (0.83) / 163.1 (1.05) / 185.7 (1.08)
Protocol Type 2, 37°C 48h, 0.95 µg/µl (only colon sample) and Protocol type 3 QIAamp DNA Mini Kit / 8 / 274.4
(1.35) / 3.0 (1.73) / 19.4
(1.88) / 20.6
(1.85)
Protocol type 3, Kit-Qiagen QIAamp DNA Mini Kit3 / 9 / 33.4
(1.99) / 4.0 (1.75) / 11.2
(1.97) / 13.6
(1.91)
Protocol type 3, Kit-Qiagen QIAamp DNA Mini Kit / 10 / 103.4
(2.00) / 12.7 (2.01) / 19.7
(2.03) / 20.6
(2.03)
Protocol Type 3, Kit-Qiagen DNeasy tissue3 / 12 / 44.0
---- / 8.8 (1.77) / 15.6
(1.74) / 18.6
(1.74)
Protocol Type 3, Kit-Macherey Nagel NucleoSpin Tissuec / 13 / 69.6
(1.43) / 10.6 (1.29) / 42.8
(1.42) / 36.9
(1.42)

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.

Supplemental Table 2: protocol type, digestion buffer composition for DNA and RNA extraction from FFPE samples for protocols not previously published.

DNA
Protocola, digestionb / Participant
Protocol Type 1, 55°C 2h or 37°C 16 h, 1.0 µg/µl
Digestion buffer: 1X PCR buffer II (without MgCl2) Applied Biosystems (Cat N° F06312 for 10X stock solution) / 2
Protocol Type 1, 55°C 16h, 4.2 µg/µl
Digestion buffer: 10 mM Tris-HCl pH 8.3, 1 mM EDTA, 0.2% Tween 20. / 3
Protocol Type 1, 56°C 16h, 1.8 µg/µl, followed by Qiagen micro spin columns
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 / 11
Protocol Type 1, 37°C 16h, 0.2 µg/µl
Digestion buffer 100 mM EDTA, 2.5% sarkosyl (Na salt of N-lauroylsarcosine. / 4
Protocol Type 2, 55°C 17h, 1.8 µg/µl
Digestion buffer: 10 mM Tris-HCl pH 8.3, 1 mM EDTA, 0.5% Tween 20. / 6
Protocol Type 2, 37°C 48h, 0.95 µg/µl (only colon sample)
Digestion buffer: 1X PCR buffer II (without MgCl2) Applied Biosystems (Cat N° F06312 for 10X stock solution). Enzyme inactivation: 10 min @94°C. / 8
RNA
Protocola, digestionb / Participant
Protocol Type 4, ON 56°C, 0.42 µg/µl
Buffer composition: 10 Mm Tris-HCl pH8, 1 mM EDTA, 2% SDS. / 4
Protocol Type 5, digestion homemade ON 55°C, 0.95 µg/µl
Buffer composition: 20 mM Tris-HCl pH8, 20 mM EDTA, 2% SDS / 8

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

Supplemental Table 3: Methods and Results of the used protocols for RNA extraction from FFPE samples

Protocol, digestiona,b / Lab / DNA Yield (mg)
(A260/280 nm )
colon / ovary / lung 1 / lung 2
Protocol Type 4 [5, 6], 55°C 45h, 6 µg/µl / 2 / 16.7 (2.0) / 1.5 (1.73) / 8.9
(1.92) / 9.2
(1.89)
Protocol Type 4 [5, 6], 55°C ON, 6 µg/µl / 5 / 22.2
(1.86) / 1.8 (1.80) / 6.1
(1.94) / 22.2
(1.99)
Protocol Type 5, digestion homemade and Trizol ON, 0.95 µg/µl / 8 / 46.9
(1.85) / 1.6 (1.62) / 23.0 (1.84) / 44.6 (1.89)
Protocol Type 5, Prot K digestion in lysis buffer (Biozym) and RNA-BEE (Bioconnect), 56°C ON, 1.5 µg/µl / 11 / 30.0
(1.99) / 3.6 (1.75) / 18.7
(1.85) / 37.5
(1.78)
Protocol Type 6, Kit-Gentra Purescript RNA 65°C 2h / 3 / 10.3 (1.7) / 0.2 (1.15) / 3.1
(1.58) / 8.2
(1.61)
Protocol Type 6, Kit-Qiagen RNeasy FFPE and Protocol Type 4 ON 56°C, 0.42 µg/µl / 4c / 11.7
(2.0) / 4.9 (1.85) / 9.3
(1.79) / 4.7
(1.87)
Protocol Type 6, Kit Roche High Pure RNA 55°C 17h / 6 / 61.0
(1.93) / 6.6 (1.78) / 19.8
(1.90) / 24.7
(1.96)
Protocol Type 6, Kit-Qiagen RNeasy FFPE 55°C 15’ / 7 / 48.4
(1.90) / 2.5 (1.79) / 39.9
(1.99) / 46.4
(1.98)
Protocol Type 6, Kit-Roche High Pure RNA 55°C ON / 9 / 33.4
(2.0) / 4.5 (1.79) / 7.3
(1.85) / 7.2
(1.87)
Protocol Type 6, Kit-Qiagen RNeasy FFPE ON 55°C / 12 / 60
(--) / 9.2 (1.89) / 40.0
(1.97) / 40.0
(1.99)

a 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.

Supplemental Table 4: Dependency of RNA yield on tumor tissue and isolation method.

RNA yield / colon cancer
> 50 µg / Participant 12 (Kit-Qiagen RNeasy), participant 6 (Kit Roche High Pure)
>= 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 RNeasy FFPE), participant 3 (Kit-Gentra Purescript RNA)
RNA yield / lung cancer
>= 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 yield / ovarian cancer
> 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)

REFERENCES

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (ed) (1998) Current Protocols in Molecular Biology. John Wiley & Sons, Inc.

2. Higuchi R (1989) Simple and rapid preparation of samples for PCR. In: PCR technology, principles and applications for DNA amplification, Ehrlich HA, (ed.), Stockton Press: NewYork, pp. 31-38.

3. Pauluzzi P, Bonin S, Gonzalez Inchaurraga MA, Stanta G, Trevisan G (2004) Detection of spirochaetal DNA simultaneously in skin biopsies, peripheral blood and urine from patients with erythema migrans. Acta dermato-venereologica 84(2):106-110.

4. Petzmann S, Ullmann R, Halbwedl I, Popper HH (2004) Analysis of chromosome-11 aberrations in pulmonary and gastrointestinal carcinoids: an array comparative genomic hybridization-based study. Virchows Arch 445(2):151-159.

5. Stanta G, Bonin S, Perin R (1998) RNA extraction from formalin-fixed and paraffin-embedded tissues. Methods in molecular biology (Clifton, NJ 86:23-26.

6. Stanta G, Schneider C (1991) RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. BioTechniques 11(3):304, 306, 308.

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