miRNA and Gemcitabine Response characteristics of the tumors are shown in Table 1. The study was approved by the Ethics Committee of Kyushu Univer- sity and was conducted according to the Ethical Guidelines for Human Genome/Gene Research enacted by the Japanese government and the Helsinki Declaration. Macrodissection After a review of representative hematoxylin and eosin- stained slides, 4–7 sections (5 lm thick) were cut from FFPE blocks for macrodissection. Adjacent normal tissues, including normal acinar tissues and adipose tissues, in the sections were removed macroscopically with a scalpel. Only the cancerous parts were used for the isolation of miRNA. TABLE 1 Correlation between median OS and clinical and patho- logical factors in patients with resected pancreatic cancer Factor Age C65 years \64 years Sex Male Female pT category pTl/pT2/pT3 p4 pN category pN0 pN1 Histological grade G1 G2 G3 Residual tumor R0 R1 Vessel invasion Positive Negative Lymphatic invasion Positive Negative Neural invasion Positive Negative Adjuvant therapy Yes No n 47 42 57 32 55 33 26 62 19 33 35 53 34 57 31 76 18 73 15 59 30 OS (95% CI) 19 (13.07–27) 19 (12–26) 23 (13.07–30.17) 14 (10–24.2) 23 (16.30–43) 12 (9.43–19) 43 (16.93–) 13.27 (11.6–22) 31 (9–) 23 (14–30.16) 12 (10–23) 26 (19–45) 12 (9–13.73) 13.73 (12–23) 27 (15–) 15 (12–24.2) 27 (14.53–) 16.93 (13.07–26) 23 (4.7–) 23 (14.53–30.17) 12 (7.7–24.2) OS overall survival, CI conﬁdence interval P value 0.675 0.7544 0.0019 0.0070 0.0804 \0.0001 0.0204 0.3004 0.6849 0.0382 2383 Isolation of miRNA miRNA was extracted from the macrodissected FFPE samples with the RNeasy FFPE kit (Qiagen, Tokyo, Japan) using a method modiﬁed from the manufacturer’s instructions. Brieﬂy, macrodissected FFPE sections were deparafﬁnized with xylene, washed with ethanol, and dried. Lysis buffer and proteinase K were added to the dried sections. The sections were incubated and Binding buffer was then added to the lysate and transferred to a gDNA Eliminator spin column (Qiagen) to remove genomic DNA. After eliminating DNA, 100% ethanol was added to the ﬂow-through. After mixing, the samples were transferred to an RNeasy MinElute column (Qiagen), which binds total RNA. After washing, the puriﬁed RNA was eluted with 50 ll of RNase-free water. Quantitative Reverse Transcription–Polymerase Chain Reaction Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed in a Chromo4 Real- Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal PCR Master Mix (Applied Biosystems, Tokyo, Japan). For the measurement of miRNA expression, we performed two-step qRT-PCR with speciﬁc primers for the indicated miRNAs (designed by Applied Biosystems) following the manufacturer’s protocol. Each sample was run in triplicate. The level of miRNA expression was calculated from a standard curve constructed by using small RNAs from CAPAN-1 cells. The expression levels of the indicated miRNAs were nor- malized to those of RNU6B. The accuracy and integrity of the PCR products were conﬁrmed with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA, USA). Statistical Analysis A data mining technique provided by the SAS Institute was used to split gene expression in high- and low-level groups on the basis of a platform that recursively partitions data according to a relationship between the X and Y val- ues, creating a tree of partitions (recursive descent partition analysis).26 By searching all possible cuts, it ﬁnds a set of cut points of X values (gene expression) that best predict the Y value (survival time). These data splits are done, recursively forming a tree of decision rules until the desired ﬁt is reached; the most signiﬁcant split is determined by the largest likelihood ratio chi-square statistic. In either case, the split is chosen to maximize the difference in the responses between the two branches of the split.