Mastercycler personal

The methodology for the cDNA synthesis was presented in a previous study [76 (link)]. Total RNA (5 ng) was reverse-transcribed into cDNA using a high capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s protocol. The compound mix was prepared in a 20 μL volume containing: 2 μL of 10× Buffer RT; 0.8 μL of 25× dNTP mix (100 mM); 2 μL of 10× RT random primers; 1 μL of MultiScribe^® reverse transcriptase; 1 μL of RNase inhibitor; 3.2 μL of nuclease-free H₂O; and 10 μL of previously isolated RNA. The reaction was processed in Mastercycler personal (Eppendorf, Hamburg, Germany) with the following thermal profile: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and 4 °C–∞ [76 (link)].
Furthermore, the obtained RNA (5 ng) was reverse-transcribed using TaqMan^® Advanced miRNA cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) according to manufacturers’ protocol. The whole procedure consisted four reactions with different profiles, which are presented in Table 2. The reactions were prepared in Mastercycler personal (Eppendorf, Hamburg, Germany).

Total RNA was extracted using the NucleoSpin^® RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) according to the protocol of the manufacturer. The RNA integrity was measured via RNA electrophoresis, and the RNA concentration was quantified using a NanoDrop^TM One (Thermo Fisher Scientific, Waltham, MA, United States). The remaining DNA was removed by using the DNAase I RNase-free kit (Ambion, Applied Biosystems, Austin, TX, United States). Contamination with DNA was discarded in the sample sets by running a control PCR with aliquots of the same RNA that had been subjected to the DNase treatment but not to the reverse-transcription step, using an Eppendorf Mastercycler^® Personal (Eppendorf AG, Hamburg, Germany). The High-Capacity cDNA Reverse Transcription kit MultiScribe^TM (Applied Biosystems, Austin, TX, United States) was used to synthesize complementary DNA (cDNA) from 2 μg of total RNA. The synthesis was made with heat denaturation of the RNA according to the instructions of the manufacturer.

PCR was performed using the general bacterial primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3′) to amplify part of the 16S rRNA gene. The PCR reaction mixture was 12.5 μL GoTaq Master Mix (Promega), 1 μL 10 μM 27F and 1492R primers (Operon), 9.5 μL Nuclease Free Water (Promega), and 1 μL of a colony resuspended in 100 μL sterilized distilled water. PCR was performed using an Eppendorf Mastercycler Personal, and the amplification conditions were one cycle of 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s; and finally one cycle of 72 °C for 7 min. Amplification of PCR reactions were confirmed using gel electrophoresis on a 0.8% agarose gel containing ethidium bromide. Successful PCRs were sequenced by Macrogen Corporation using the 27F primer using the Applied Biosystems 3730xl DNA analyzer. Quality control for sequences was performed using DNA Baser (www.DnaBaser.com), in which ends were trimmed until there were more than 75% good bases (defined by having a QV score of higher than 25) in an 18-base window. Identification of phylogenetic neighbors and calculations of pairwise sequence similarity were done using the EZTaxon server (http://www.eztaxon.org). 98.7% similarity by 16S was used to determine species level cutoff using a previously suggested level [32 (link)].