Supplementary Information (doc 28K)

Supplementary Information
Nucleic acids extraction, RNA purification and cDNA synthesis – detailed description
Total nucleic acids were extracted the following way: 0.5 g of frozen soil sample was placed in a
Lysing Matrix E tube (MP). 375 µL SPB (10 mM sodium phosphate buffer, pH 8.0) and 125 µL TNS
buffer (500 mM Tris HCL, 100 mM NaCl, 10% sodium dodecyl sulphate) were added to the tube; both
solutions taken from Henckel and colleagues (1999). Then, 500 µL of Tris EDTA saturated phenol
solution (pH 8.0; Fluka) were added to the tube and it was processed immediately in a FastPrep®-24
bead beating instrument for 30 s at 6.5 m s-1. The tube was immediately transferred to ice and chilled
for a minute and then centrifuged for 5 min at 14,000 rpm. The supernatant (including phenolic
phase) was transferred to a 2 mL tube which was kept on ice and the beads tube containing the soil
sample was retained. This process was repeated twice more with fresh aliquots of buffers and phenol
solution. At the third time, the supernatant (excluding the phenolic phase) was distributed between
the two 2 mL tubes used in to store the supernatant in the previous steps and the beads tube was
discarded. Approx. 800 µL phenol/chloroform/isoamylalcohol (25:24:1; Fluka) were added to each of
the tubes containing the extractant to a 2 mL total volume. The phases were homogenised by mixing
and the tubes were centrifuged for 3 min at 14,000 rpm. The supernatants were transferred from
each tube to fresh 2 mL tubes, and 1 volume of chloroform/isoamylalcohol (24:1; Sigma-Aldric) was
added to each. The phases were homogenised again by mixing and the tubes were centrifuged for 3
min at 14,000 rpm. The supernatants were transferred from each tube to Non-Stick RNase-free
Microfuge Tubes (Ambion). To each tube 1 µL of glycogen (5 mg mL-1; Ambion) and 1.5 mL PEG
precipitation solution (20% polyethylen glycol, 2.5 M NaCl) were added and the tubes were
centrifuged for 90 min at 14,000 rpm and 4 oC. After centrifugation, the entire supernatant was
discarded and the pellets were washed with ice cold 75% ethanol and centrifuged again for 20 min at
14,000 rpm and 4 oC. The entire supernatant was removed again, the pellets were dried under a
stream of filtered N2, then resuspended in 100 µL of low TE buffer (10mM Tris, 1mM EDTA) and the
content of the two tubes was combined. In cases when the extractant had colour it was further
purified using MicroSpin S-200 HR (GE Healthcare). 10 µL of the extractant was diluted in 90 µL of
low TE buffer and was used as DNA template. For synthesis of complete cDNA, 50 µL of nucleic acids
extractant were digested with TURBO™ DNase (Ambion) and later purified using RNeasy® MinElute®
Cleanup Kit (Qiagen) and quantified using a NanoDrop 1000 spectrophotometer (Thermo). Complete
DNA removal was verified by failure to obtain PCR amplification product with the purified RNA
template using the conditions described below. Complete cDNA was synthesized using ImProm-II™
Reverse Transcriptase (Promega) by the following procedure: 100 ng purified RNA and 0.5 µg/µL of
random hexamer primers were incubated at 70°C for 5 min followed by 5 min chilling on ice. The
following mixture was then directly added to the tubes: 4 µL of 5× ImProm-II™ Reaction Buffer, 3 mM
MgCl2, 1 mM of each dNTP, 20 µg of bovine serum albumin (BSA) (Roche), and 20 U of Recombinant
RNasin Ribonuclease Inhibitor (Promega). The reaction tubes were incubated at 25°C for 5 min
followed by 52°C for 1 h, and then denaturation at 70°C for 15 min. The resulting cDNA product was
used directly as a template for consecutive PCR reactions. PCR amplifications were performed
identically for both DNA and cDNA templates.
Henckel T, Friedrich M, Conrad R. (1999). Molecular Analyses of the Methane-Oxidizing Microbial
Community in Rice Field Soil by Targeting the Genes of the 16S rRNA, Particulate Methane
Monooxygenase, and Methanol Dehydrogenase. Appl. Environ. Microbiol. 65:1980-1990.