Rna plant kit

Total RNA for first-strand cDNA synthesis was isolated from cultures of ATCC 1015. The biomass was grown under manganese paucity in 500 mL Erlenmeyer flasks (VWR International Kft., Debrecen, Hungary) with 100 mL of growth medium (2.50 g (NH₄)₂SO₄; 0.15 g KH₂PO₄; 0.15 g NaCl; 2.25 g MgSO₄·7H₂O; 1.50 mg Zn²⁺; 140 g D-glucose; 0.10 mg Fe²⁺ and 0.06 mg Cu²⁺, per litre) (i.e., no MnCl₂ added) in a rotary shaker (Infors AG, Basel, Switzerland) at 250 rpm at 30 °C. Total RNA was isolated using the RNA Plant kit (Macherey-Nagel GmbH & Co., KG, Düren, Germany). First-strand cDNA was synthesized from a total RNA template with Oligo(dT) as the primer using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA). First strand cDNA was subsequently used as the template for the PCR reaction, and the cycling conditions after initial denaturation at 95 °C (3 min) were as follows: 35 cycles of 95 °C for 30 s, 54 °C for 1 min, and 72 °C for 0.5–1 min, followed by one post-cyclic elongation at 72 °C (5 min). The downstream sequencing procedure after cloning of the RT-PCR amplification was the same as described for gDNA (above). All RT-PCR experiments were performed in triplicate, starting with biomass from three independent liquid cultures.

DNA and RNA was isolated from a pinhead amount of alga culture growing on agar plate using a DNA plant kit and RNA plant kit, respectively (Macherey Nagel, Germany) according to the manufacturer’s recommendation. The isolation includes 15 min on-column of enzymatic RNase and DNase treatment, respectively. DNA from lichen samples was isolated using the Wizard Magnetic 96 DNA Plant System kit (Promega, USA) from about 100 mg of dry lichen thalli in 50 μl of sterile water. The iScript cDNA synthesis kit (Bio-Rad, USA) was used for cDNA synthesis.

After germination of spores, pieces of the mycelium were directly used or grown at 27° and constant light on M2 medium for 13 – 16 days (middle-aged) or 21 – 24 days (senescent), depending on the lifespan of the specific individual, to obtain cultures of specific age. A piece of the growth front was subsequently spread on a fresh M2 plate covered with cellophane (BioRad Cat# 1650963) and grown for 3 days. RNA was extracted with RNA-Plant kit (Machery-Nagel Cat# 740.949.250) and cDNA synthesis was performed using iScript kit (BioRad Cat# 170-8891). After dilution of cDNA to a concentration of 10 ng/µl, 20 ng was used per qRT-PCR reaction (IQ SybrGreen SuperMix, BioRad cat# 170-8882). The primers summarized in
Table 1 were used to perform the qRT-PCR with three technical replicates per sample. A specific culture was compared to the mean CP of the juvenile cultures. Relative expression was normalized to
PaPorin with the following formula
^{26 (link)}.
$\mathit{\text{ratio}}=\frac{{\left(E_{\mathit{\text{target}}}\right)}^{\Delta {\text{CP}}_{{\mathit{\text{target}}}^{\left(\mathit{\text{control–sample}}\right)}}}}{{\left(E_{\mathit{\text{Porin}}}\right)}^{\Delta {\text{CP}}_{{\mathit{\text{Porin}}}^{\left(\mathit{\text{control–sample}}\right)}}}}$
E = PCR-Efficiency; CP = crossing point