RNA extraction
For RNA extraction, the whole ventriculus, foregut, midgut and hindgut
of each specimen was eviscerated under the binocular magnifier from the
parental adults (11 samples), larvae (7 samples) and teneral adults (8
samples) of Ips typographus . Pupae were not analyzed as the
softness of their bodies impeded such manipulation. RNA was extracted
using the Nucleospin RNA plant kit (Macherey-Nagel). The intestines were
eviscerated directly into lysis buffer RA1 supplemented with 1%
β-mercaptoethanol and then kept in the freezer at -80°C until needed.
Samples in lysis buffer were then homogenized using Lysing Matrix A (MP
Biomedicals) on FastPrep-24™ 5G Instrument (Irvine, California, USA) for
30 s at 5.5 m/s. The DNA digestion step was omitted from the protocol.
The DNA digestion was then performed with TURBO DNA free kit
(Invitrogen). The absence of residual DNA in the samples was verified by
PCR amplification of ITS and 16S regions and visualization of the
reaction products on the agarose gel. The purity of isolated RNA was
checked by Nanodrop (NanoDrop™ 2000c, ThermoFisher scientific). Samples
with A260/A280 and A260/A230 lower than 1.8 were repurified by
isopropanol precipitation with 3 M sodium acetate, pH 5.2. RNA
concentration was measured on Qubit 2.0 Fluorometer (Thermo Fisher
Scientific, Waltham, Massachusetts, USA) using Qubit dsRNA BR Assay Kit.
Quality of isolated RNA was examined on Bioanalyzer using RNA 6000 Pico
Kit (Agilent).