Supplementary Material and Methods (including one reference)
Total DNA was extracted using the commercial GentraPuregene Tissue Kit (Qiagen) and according the « DNA purification from fixed tissue » instructions of the manufacturer. Sample disruption and homogenization was achieved by combining the chemical solution of the kit and mechanical grinding with single-use beads (Lysing Matrix A, MP Biomedicals). Briefly, a small piece of each frozen sample was excised using a razor blade (on dry ice), transferred in an individual Lysing Matrix Tube filled with 300 µL of Cell Lysis Solution, weighted and incubated for 15 min at 65 °C. Homogenization was performed using a FastPrep120 instrument (MP Biomedicals). Skin homogenization cycle ran twice at maximum speed (v = 6) during 20 sec. A complementary step of 20 sec at v = 4 was added for tail samples. The following treatments were exactly as described by the manufacturer including proteinase K digestion, RNAse treatment, protein removal and DNA precipitation.
DNA concentration and purity (absorbance ratios A260/280 and A260/230) were determined with a spectrophotometer NanoDrop 1000 system (ThermoFischer Scientific) and the ND-1000 3.3 associated software (NanoDrop Technologies). DNA extraction yield squares with the quantity of nucleic acids extracted from a given sample tissue relating to its weight (µg DNA / mg tissue). DNA integrity was visualized by electrophoresis on a 0.8 % agarose gel and standard ethidium bromide staining. Images were captured using PerfectImage software (ClaraVision).
Qualitative DNA integrity was assessed by the capability of amplifying by PCR the mitochondrial gene Cytochrome c Oxydase I (COI or CoxI). Using the “Primer3” program (http://biotools.umassmed.edu/bioapps/primer3_www.cgi#disclaimer (Rozen and Skaletsky 1999)), we designed eight primers on the sequence AY172581 (bases 5323 to 6867) of Genbank Nucleotide database (http://www.ncbi.nlm.nih.gov/genbank/): 100F (5-cttcgggaactgacttgtacc-3’), 100R (5’-ggagtagaaatgatggaggaagc-3’), 149F (5’-ttacaccacatgaaatacagtctcc-3’), 149R (5’-gcagtcattctaggttagttgagg-3’), 499F (5’-tcttaattcttccagggtttgg-3’), 499R (5’-tttgctcatgtgtcatttaggg-3’), 1F (5’-ccattttacctatgctcgtaaacc-3’), 1R (5’-accagttgtagggggttcg-3’). Those primers were combined to obtain five different amplicons sizing from 104 bp to 1497 bp: C100 (104 bp) with 100F + 100R, C240 (240 bp) with 149F + 1R, C500 (499 bp) with 499F + 499R, C1000 (1002 bp) with 100F + 499R and C1500 (1497 bp) with 1F + 149R.
Qualitative PCR assessment of DNA
PCR were carried out in 10 µL reaction volume. The reaction mix contained 10 ng of DNA, 1 µg/mL yeast tRNA (Roche), 1 µL of 10 x reaction buffer (Invitrogen), 0.5 µM of each primer, 40 µM of each dNTP (Invitrogen), 2.5 mM MgCl2 (Invitrogen) and 0.5 U of Taq DNA Polymerase (Invitrogen). The thermal conditions were an initial step at 95 °C for 10 min followed by 30 three-steps amplification cycles (95 °C, 30 s ; 60°C, 30 s ; 72 °C, 30 s for amplicons shorter than 500 bp or 1 min for amplicons longer than 500 bp), and ending with an extension step at 72 °C for 10 min. Negative controls were
PCR fragments were resolved by electrophoresis on 1 % agarose gels (amplicons longer than 500 bp) or on 3 % Low Melting Nusieve GTG agarose (Tebu-Bio) gels for fragments shorter than 500 bp. Migration conditions were: 135 V in 1 x TBE buffer at room temperature or 4 °C (for low melting gels). Visualization and image capture were performed using standard ethidium bromide staining and the PerfectImage software.
Quantitative PCR assessment of DNA
The ability to quantitatively amplify the locus Cox1 was estimated using EvaGreen based qPCR assay with three different short amplicons ranging from 104 to 240 bp: C100 and C240 were as described above, C149 was a 149 bp fragment obtained with primers 149F and 149R.
Reactions were done in Fast Optical 96 wells plate (Applied Biosystems) sealed with Optical Adhesive Cover and run on StepOne Plus system (Applied Biosystems). Assays were performed in a 10 µL reaction volume containing 2.5 µL of DNA template, 0.5 µM of each primer and 5 µL of 10 x Sso Fast EvaGreen Low Rox Supermix (Biorad). Thermal conditions were initial denaturation and enzyme activation step at 95 °C for 3 min followed by 40 two-steps amplification cycles (95 °C,15 s ; 60 °C, 1 min) and ending with a dissociation step (95 °C, 15 s ; 60 °C to 95 °C with + 0.5 °C / min ; 95 °C, 15 s).
All samples (T1, 2h, 4h, 6h and 24h) of the three specimens (A, B and C) treated in a same kinetic of treatment were processed on the same run. Comparisons between samples were made for 2.5 ng of DNA. PCR efficiencies were determined, each time, with serial dilutions of each T1, 4 h and 24 h DNA templates prepared in UltraPure DNAse/RNAse free water (Gibco) in presence of 1 µg/mL yeast tRNA. Dilutions were as follow (total DNA per reaction):
- skin: 10 ng/μL (25 ng) ; 1 ng/μL (2.5 ng) ; 100 pg/μL (250 pg) ; 10 pg/μL (25 pg),
- tail: 5 ng/μL (12.5 ng) ; 1 ng/μL (2.5 ng) ; 100 pg/μL (250 pg) ; 10 pg/μL (25 pg).
On average (n>30), primer pairs efficiencies were determined as C100: 98.54 % 4.715, C149: 94.74 % 3.886 and C240: 91.39 % 8.031.
Negative controls were made in absence of template and/or absence of primers.
Data were collected and analyzed using the StepOne Software v2.0 associated with its dedicated detection system. Each sample tested is defined by a Cq (quantification Cycle) value representing its amplification level.
Rozen S, Skaletsky HJ (1999) Primer3 on the WWW for General Users and for Biologist Programmers. Meth Mol Biol 132:365-86