Applied biosystems 7500 thermocycler

Total RNA was extracted from rice leaves using Trizol (Invitrogen) and the Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s instructions. The FastKing gDNA Dispelling RT SuperMix (TIANGEN Biotech Co., Beijing) was used for the synthesis of first-strand cDNA. qRT-PCR using the primers listed in Supplementary Table 8 was carried out with SuperReal PreMix Plus (SYBR Green) (TIANGEN Biotech Co., Beijing) following the manufacturer’s instructions. qPCR was performed with an Applied Biosystems 7500 thermocycler (Thermo Fisher Scientific, USA) under the following cycling conditions: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. The expression of rice Actin1 (LOC_Os03g50890) was used as an internal control to normalize target gene expression. The relative expression of each gene was calculated based on the 2^−△△CT method^{96 (link)}.

Using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), total RNAs from cells were extracted and then reverse transcribed by PrimeScript RT Reagent kit (TaKaRa, Tokyo, Japan). The mRNA level of PABPC1 was tested by RT-qPCR with the SYBR Premix Ex Taq (TaKaRa) on the Applied Biosystems 7500 Thermocycler (Thermo Fisher Scientific) according to the manufacturer’s protocols. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the control. Primer sequences for RT-qPCR were:
PABPC1 forward: 5′-CAGAGAATGGCAAGTGTACGAGC-3′,
PABPC1 reverse: 5′-GCTAGGAGGATAGTATGCAGCAC-3′;
GAPDH forward: 5′-TGTGTCCGTCGTGGATCTGA-3′,
GAPDH reverse: 5′-CCTGCTTCACCACCTTCTTGA-3′.

BMFS samples were analyzed at the CDC according to the WHO algorithm (WHO 2015 ) and ITD for SL1, SL2, SL3, WPV1, WPV3, panPV, and panEV (Fig. 1, A5). Virus isolation on two cell lines (L20B and RD cells) was performed prior to detection of PV in the cell culture supernatant (virus isolate) by ITD using rRT-PCR (Poliovirus ITD 4.0/4.1 rRT-PCR Kit [CDC, Atlanta, GA, USA]) (WHO 2015 ). Primers and probes used for detection of SL1, SL2, and SL3 are shown in the Online Resource (Table S1) (Kilpatrick et al. 2009 (link)). Primers and probes used for detection of WPV1 and WPV3 were in the Poliovirus ITD 5.0 rRT-PCR Kit, as described in Gerloff et al. (2018 (link)). Real-time RT-PCR was performed using an Applied Biosystems® 7500 thermocycler (Applied Biosystems, Foster City, CA, USA), and the programs used are shown in the Online Resource (Table S2).

The level of expression of neurotrophic factors (BDNF, GDNF) and their key receptors (TrkB, GFRα1) in the brain tissues of animals was assessed by real-time PCR analysis.
Total RNA from the cerebral cortex and hippocampus of the mouse brain was isolated by phenol–chloroform extraction using ExtractRNA (Eurogen, Russia) according to the manufacturer’s protocol. MMLV RT kit and random primer (Eurogen, Russia) were used for reverse transcription.
Real-time PCR was performed using a ready-made reaction mixture qPCRmix-HS SYBR+LowROX (Eurogen, Russia).
The following sequences of primer pairs were used:
Oaz1_fw 5′-AAGGACAGTTTTGCAGCTCTCC-3′;
Oaz1_rv 5′-TCTGTCCTCACGGTTCTTGGG-3′;
BDNF_fw 5′-CCCAACGAAGAAAACCATAAGGA-3′;
BDNF_rv 5′-CCAGCAGAAAGAGTAGAGGAGGCT-3′;
GDNF_fw 5′-CCTTCGCGCTGACCAGTGACT-3′;
GDNF_rv 5′-GCCGCTTGTTTATCTGGTGACC-3′;
TrkB_fw 5′-TTTCCGCCACCTTGACTTGTCT-3′;
TrkB_rv 5′-GTCGGGGCTGGATTTAGTCTCC-3′;
GFRα1_fw 5′-TGTCTTTCTGATAATGATTACGGA-3′;
GFRα1_rv 5′-CTACGATGTTTCTGCCAATGATA-3′.
qPCR conditions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 60 s on an Applied Biosystems 7500 thermocycler (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).
The results were processed by the ΔΔCt method using samples obtained from the control (not injected) group of animals, in which the expression level was taken as one. Oaz1 was used as a reference gene.