Thermal cycler dice real time system 3

To isolate the total RNA, we used the TriZol reagent (Life Technologies, Gaithersburg, MD, USA) and obtained cDNA using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA). For PCR, we used Blend Taq DNA polymerase (Toyobo, Osaka, Japan) with primers targeting DR5, c-FLIP(L), and actin as mentioned in our previous studies [37 (link)]. For qPCR, we utilized the SYBR Fast qPCR Mix (Takara Bio Inc., Shiga, Japan) and reactions were performed on the Thermal Cycler Dice^® Real Time System III (Takara Bio Inc., Shiga, Japan). The following primers were used for the amplification of DR5, c-FLIP(L), and actin as described as our previous study [37 (link)]. We used actin as a reference gene to calculate the threshold cycle number (Ct) of the DR5 gene and reported the delta-delta Ct values of the genes.

To isolate the total RNA, we used TriZol reagent (Life Technologies, Gaithersburg, MD, USA), and obtained cDNA using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA). For PCR, we used Blend Taq DNA polymerase (Toyobo, Osaka, Japan) with primers targeting DR5, Cbl, survivin and actin as mentioned in our previous studies [46 (link),47 (link)]. For qPCR we utilize SYBR Fast qPCR Mix (Takara Bio Inc., Shiga, Japan) and reactions were performed on Thermal Cycler Dice^® Real Time System III (Takara Bio Inc., Shiga, Japan). The following primers were used for the amplification of DR5 and actin as described as our previous study [23 (link)]. We used actin as a reference Gene to calculate the threshold cycle number (Ct) of DR5 gene and reported the delta-delta Ct values of the genes.

The total cellular RNA was isolated from mouse gastrocnemius muscle using the Sepasol-RNA I Super G (Nacalai (Japan)) according to the manufacturer’s instructions. cDNAs were synthesized using the SuperScript IV first-strand synthesis system for RT-PCR (Termo Fisher Scientific (MA, USA)). PCR was carried out using TB Green Premix Ex Taq II (Takara Bio (Kyoto, Japan)) and specific primers (Termo Fisher Scientific) (5´-TGTGATGGAGTATGCCAACG-3´ and 5´-CTCCAGAGCTGACACAATCT-3´ for Akt2, and 5´-ATGAAGATCAAGATCATTGCTCCTC-3´ and 5´-ACATCTGCTGGAAGGTGGACAG-3´ for β-actin) with Thermal Cycler Dice Real Time System III (Takara Bio) according to the manufacturer’s instructions. The mRNA levels were determined by the comparative Ct method followed by normalization with the β-actin mRNA level in each cDNA sample.

Quantitative RT-PCR (qRT-PCR) for annotated genes located in the region of qTSN12.2 was performed. IR 64 and IR 64-NIL12 were grown in an experimental paddy field of NICS, Ibaraki, Japan (36°00′N, 140°01′E) in 2016. Note that increased TSN in IR 64-NIL12 was stably observed also in this experimental paddy field (see Supplementary Table S2). Three developing panicles (2–5 mm) were collected as one biological replicate from a plant at panicle initiation stage, and four biological replicates (four plants) were prepared for this expression analyses in both genotypes. Total RNA was extracted by using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was used for the reverse transcriptase reaction (Prime Script RTase, Takara Bio Inc., Otsu, Japan). qRT-PCR reactions were carried out with 1.5 μl cDNA mixtures under a Thermal Cycler Dice Real Time System III (Takara Bio). The expression level of annotated genes was normalized to the expression of a household gene, ubiquitin (Os01g22490), in the developing panicles (Fujita et al., 2013 (link)). The expression of each gene was compared between IR 64 and IR 64-NIL12 using the Δ
Δ
C_t method (Livak and Schmittgen, 2001 (link)). All statistics were performed at the Δ
C_t stage with the t-test.

Total RNA was isolated using TriZol reagent (Life Technologies, Gaithersburg, MD, USA), and cDNA was prepared using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA). For PCR, we used Blend Taq DNA polymerase (Toyobo, Osaka, Japan) with primers targeting DR5, c-FLIP, Mcl-1 and actin. For qPCR, SYBR Fast qPCR Mix (Takara Bio Inc., Shiga, Japan) was used, and reactions were performed on a Thermal Cycler Dice^® Real Time System III (Takara Bio Inc., Shiga, Japan). We calculated the threshold cycle number (Ct) of each gene using actin as the reference gene, and we reported the delta-delta Ct values of the genes. The used primers were referred to in previous studies [33 (link)].

Total RNAs were obtained by CellAmp™ Direct RNA Prep Kit for RT-PCR (TaKaRa Bio Inc., Shiga, Japan) from C6/36 cells in a 96-well plate inoculated with EVs derived from BHK/JM-PnL and BHK/DN-PnL cells. Total RNAs of the EVs treated with RNase A in the presence/absence of Tx-100 were isolated by a microRNA Extractor^® Kit for Purified EV (FUJIFILM Wako Pure Chemical Corporation). JM-PnL and DN-PnL replicon genomes were quantified by RNA-direct™ SYBR^® Green Realtime PCR Master Mix (TOYOBO). Primers used for JM-PnL were sense, 5′-ccctcagaaccgtctcggaa-3′, and anti-sense, 5′-ctattcccaggtgtcaatatgctgt-3′, and primers used for DN-PnL were sense, 5′-agttgttagtctacgtggaccga-3′, and anti-sense, 5′-cgcgtttcagcatattgaaag-3′. The real-time PCR was carried out by Thermal Cycler Dice Real-Time System III (Takara Bio Inc.). The in vitro-transcribed JM-PnL and DN-PnL were used as standard.

BLV-infected cattle with high proviral load (HPL) in blood were selected for this study. It was reported that BLV-infected cattle with HPL in blood were considered as cattle at high risk to be BLV spreaders and might be one of the factors of disease progression [20 (link)]. BLV proviral load was measured by using 100 ng of WBC DNA by real-time PCR (qRT-PCR). The amplification was carried out in a reaction mixture containing 12.5 µL of 2× CycleavePCR Reaction Mix (CY510, Takara Bio), 5 µL of probe/primer/positive control for BLV (CY415, Takara Bio), 5 µL of a template DNA sample, and PCR grade water to increase the volume up to 25 µL. For the proviral quantification, BLV tax gene was used as a control from the kit (CY415, Takara Bio) and BLV proviral DNA was measured by a Thermal Cycler Dice Real Time System III (TP970, Takara Bio) according to the manufacturer’s instructions. After the measurement, BLV proviral copies of > 5000/100 ng of WBC DNA was considered HPL in BLV-infected cattle (Table 1). Hematology test, detection of serum antibodies against BLV, detection of BLV provirus, and measurement of BLV proviral load were conducted by the Gifu Central Livestock Hygiene Service Center (Gifu, Japan).