User bulletin 2

For each sample and for each miRNA, 5 ng of total RNA was converted to cDNA by TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) with the miRNA specific primer contained in the TaqMan MicroRNA assays (Applied Biosystems). MiRNA expression was evaluated in 10 μL total volume by quantitative RT-PCR following the manufacturer protocol in the presence of 1× TaqMan Universal Master Mix (Applied Biosystems) on a 7900HT SDS instrument (Applied Biosystems). Expression differences of miRNAs among samples were determined by the comparative method according to User Bulletin #2 (Applied Biosystems) using the noncoding RNA RNU44 and RNU6B as reference gene.

The qRT-PCR was performed as previously described [12 (link), 13 (link)]. All experiments were performed in triplicate. Relative fluorescence was calculated using the ΔΔ-CT method, as outlined in User Bulletin 2 (PE Applied Biosystems, Darmstadt, Germany). The statistical significance of qPCR values at different time points was assessed by Student's paired t-test.

Statistical significance was analysed using the F test, which was used for testing the homogeneity of variances, followed by the Student’s t test (P > 0.2 by the F test) or the Aspin-Welch test (P ≤ 0.2 by the F test). Student’s t tests and Aspin-Welch tests were conducted at the two-tailed significance levels of 5% (0.05). Cumulative survival curves were constructed using Kaplan-Meier survivorship methods. Differences between the curves were tested for significance by the log-rank test. All data were presented as the means and SD, except for qPCR data generated using the comparative C_T (ΔΔC_T) method (User Bulletin #2, Applied Biosystems). For the comparative C_T method, the ΔC_T value was determined by subtracting the average internal control C_T value from the average target C_T value. The calculation of ΔΔC_T involves subtraction by the ΔC_T calibrator value. The range given for each mRNA level relative to the control was determined by evaluating the expression: 2^−ΔΔCT with ΔΔC_T + s and ΔΔC_T − s, where s = the standard deviation of the ΔΔC_T value.

Messenger RNA expression of the major RASSF1 variants RASSF1A, RASSF1B and RASSF1C was determined in 14 xenografted PDAC and 8 PDAC cell lines. RNA samples were retrotranscribed to cDNA using the First Strand cDNA Synthesis Kit (Roche). A reverse transcriptase minus cDNA was prepared for each sample as a control. QRT-PCR was carried out as previously described [36 (link)] in 25 μl total volume containing 4 ng of cDNA, 1x Power SYBR Green I Master Mix (Applied Biosystems), 400 nM of each primer. After a starting denaturation for 10 min at 95 °C, 45 PCR cycles (15 s 95 °C and 1 min 60 °C) have been performed on ABI PRISM 7900HT SDS instrument (Applied Biosystems). The relative expression level was calculated using transcript level of RPLPO as reference gene and the standard (=1) was the average of the levels of expression of all samples. QRT-PCR data analysis was performed according to the comparative method following the User Bulletin #2 (Applied Biosystems).

Quantitative real-time PCR analysis of Igf-1, Igf-1r, and beta actin (Actb) transcripts was performed using TaqMan assays (Rn00710306_m1, Rn00583837, Rn00667869_m1, respectively) the TaqMan Universal PCR Master Mix (Applied Biosystems), and the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Life Technologies, Warrington, UK). Ct values were analyzed with the standard curve method (User Bulletin #2, Applied Biosystems) and normalized to housekeeping gene Actb.

After BABA or water drenched for 3 days, primary leaves were inoculated with 6 apterous aphids as follows: to confine aphids movement, a 2.5 cm×2.5 cm piece of sticky plastic sheet with a 1.5 cm-diameter hole in the centre was sticked to the upper side of the leaflet of each plants and 6 apterous aphids were placed in each hole with a moist brush slightly, and then covered the hole with organdy fabric [30] (link). After 12, 24, 36 and 48 h, the attacked sites were cut by surgical scissors and aphids were removed quickly using a moist brush. Leaf samples at 0 h were not inoculated with aphids and also brushed when collected. Aphid- or mock-treated leaves for each of six plants were pooled together (three replications for each treatment), respectively, and immediately frozen in liquid nitrogen. All of the leaves were stored at −80°C before RNA isolation. All gene-specific primers were described in Table 1. Relative expression levels at each time point were calculated from cycle threshold (CT) values according to the ΔCT method (Applied Biosystems User Bulletin#2) using the soybean actin gene as a reference[31] (link)–[43] (link).