Abi viia 7 thermocycler

Nitrogen fixation potential of cyanospheres and bulk soil communities was evaluated using quantitative real-time PCR (qPCR) to quantify the gene copy number of 16S rRNA (a proxy for bacterial biomass) and nifH (a proxy for nitrogen fixation capacity) genes of the appropriate community DNA extracts, as previously described [7 (link), 39 (link)]. For 16S rRNA gene quantitation, a universal (bacterial/archaeal) primer set (338 F 5'- ACTCCTACGG GAGGCAGCAG -3', 518 R 5'- GTATTACCG CGGCTGCTGG -3') [57 (link)] was used. For nifH quantitation, a standard primer set for nifH (PolF 5'- TGCGAYCCSA ARGCBGACTC-3', PolR 5’-ATSGCCATCA TYTCRCCGGA-3') [58 (link)] was used. The PCR reactions were performed in triplicate using PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, USA) in an ABI ViiA 7 thermocycler (Applied Biosystems, Foster City, CA, USA) under conditions previously published [59 (link)]. Nitrogen fixation potential for each bundle or bulk soil community assessed as the ratio of nifH to16S gene copy numbers detected. Ratios were log transformed to comply with normality and variance homogeneity before testing for significance with a one-way ANOVA using R [54 ]. 16S rRNA and nifH gene quantitation were also performed on DNA extracts from cotton thread controls to determine if contaminants had an impact on nitrogen fixation potentials.

Total RNA was purified from HDMECs and HeLa cells using the RNeasy kit, according to the manufacturer’s instructions (Qiagen, Manchester, UK). RNA quantity and quality were determined using a NanoDrop 1000 Spectrometer (Thermo Fisher Scientific). Then, 1 µg of RNA was reverse-transcribed to cDNA using the reverse transcriptase Superscript III in addition to oligo dT, random hexamers, dNTP mix, and RiboLock (Life Technologies, Paisley, UK). To set up a 25 µL reaction for qPCR, 10ng of cDNA was mixed with RNase free water, 2× Power SYBR Green (Life Technologies, Paisley, UK) and forward and reverse primers to a final concentration of 400 nM (Life Technologies, Paisley, UK).
Primer sequences (5’ to 3’):
β-ACTIN Fwd GATGAGATTGGCATGGCTTT; β-ACTIN Rev CACCTTCACCGTTCCAGTTT. MEF2A Fwd CAGGGAGTTCACTGGTGTCC; MEF2A Rev CTTGGTGGTCTCTGAGGAGC. MEF2B Fwd GTTCACCAAGCGGAAGTTCG; MEF2B Rev GCATACTGGAAGAGGCGGTT. MEF2C Fwd CGAGATGCCAGTCTCCATCC; MEF2C Rev GTGAGCCAGTGGCAATAGGT. MEF2D Fwd AGGAAGAAGGGCTTCAACGG; MEF2D Rev GTCGGTACTTGTCCTCCAGC.
qPCR was performed on an ABI ViiA 7 Thermocycler (Applied Biosystems) with the following parameters: 50 °C for 2 min; 95 °C for 10 min; (95 °C for 15 s and 60 °C for 1 min) × 40 cycles. For each sample, the average cycle threshold (CT) value was normalised to β-ACTIN and then compared to the relevant control sample using the comparative CT (2-ΔΔCT) method.