Abi 7900 fast sequence detection system

Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and stored at -80°C. The quantitative real-time PCR was performed using a 2-step method accord-ing to manufacturer instructions (Takara, Shiga, Japan). Real-time PCR was conducted using the ABI 7900 fast sequence detection system (Applied Biosystems) with the SYBR green fluorescent dye. The cycling parameters were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 1 min, and elongation at 72°C for 30 s. The human b-actin transcript was used as an internal reference to control for variations in the total mRNA quantity of each sample. Each RNA sample was analyzed in triplicate using the following primers: Human miR-196a primers: forward 5'-GAGGCGTGGCAGACTATGC-3'; reverse 5'-CTTGTACTCCGTCAGCGTGA-3'; human miR-146a primers: forward 5'-TGTAACCAGAG AGCGGGATGT-3'; reverse 5'-TTTTGGCATAACTAAGGCCGAA-3'; human b-actin primers: forward 5'-CACTCTTCCAGCCTTCCTTC-3'; reverse 5'-GGATGTCCACGTCACACTTC-3'.

RNA isolation and qPCR have been described before [15] . Briefly, total RNA was reverse-transcribed into single-strand complementary DNA (cDNA) using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Analysis of the results was performed in duplicate with an ABI 7900 Fast Sequence Detection System using TaqMan gene expression assays and Universal PCR Master Mix according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA) as described before [15] . The TaqMan assays were UCN3 (Assay ID: Hs00846499_s1), GUSB (Assay ID: Hs00939627_m1), RPL134 (Assay ID: Hs03043885_g1), and HPRT1 (Assay ID: Hs99999909_m1). The human GUSB, RPL134, Relative quantities of transcripts were calculated using the SDS 2.3 Manager data assist v2.0 software and the 2 -DDCt method as described before [15] [16] [17] , but replacing the biological control by the average Ct values for UCN3 and the endogenous controls as obtained from the whole tissue sample group.

Total RNA was extracted with the help of the TRIzol reagent (Invitrogen) and stored at -80°C. Quantitative real-time PCR (qRT-PCR) was conducted in a 2-step fashion as per the manufacturer's instructions (Takara). The testing of real-time PCR required the use of the ABI 7900 fast sequence detection system (Applied Biosystems) as well as the SYBR green fluorescent dye. The following PCR was used: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and finally an elongation step at 72°C for 30 seconds. To control for variations in the total quantity of miRNA in each sample, an internal reference (the U6 snRNA) was used. The differential expression was evaluated with the 2-(∆∆Ct) method. Each RNA sample was analyzed 3 times with the following primers: 1) human Notch1 primers: forward 5′-GAGGCGTGGCAGAC-TATGC-3′; reverse 5′-CTTGTACTCCGTCAGCGTGA-3′; 2) human miR-146a primers: forward 5′-TGTAACCAGAGAGCGGGAT-GT-3′; reverse 5′-TTTTGGCATAACTAAGGCCGAA-3′; 3) human U6 snRNA primers: forward 5′-CTCGCTTCGGCAGCACA-3′; reverse 5′-AACGCTTCACGAATTTGCGT-3′.