miRNA Microarray
Total RNAs of the two littermate piglets were pooled for the microarray analysis. The pooled samples were named as EHL1–3 (n
=3) and LW1-3 (n=3).
The pig microRNA microarray was obtained from LC Sciences (Houston, USA) and contains 238 unique probes that were complementary to all mature miRNAs of pig in miRBase release 16.0. The assay started from 5 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore, USA) and the small RNAs (<300 nt) isolated were 3′-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies, USA). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target miRNAs (from miRBase, release 16, 238 probe sets) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent (PGR) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. 100 µL 6×SSPE buffer containing 25% formamide was used for hybridization at 34°C. After hybridization, fluorescence labeling using tag-specific Cy5 dyes was used for detection. Hybridization images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS (locally weighted regression) function. The three samples of LW piglets were used as control group to analysis the different expression between the two pig lines. The differences between the two groups were analyzed using SAM (Significance Analysis of Microarrays) method with SAMR software version 3.02 [42]. The FDR (False Discovery Rate) and fold change were calculated. miRNAs with FDR <15% were considered to be differentially expressed miRNAs. All the microarray data were MIAME compliant and have been deposited in GEO (accession number GSE33523).
Real-time qPCR Confirmation of Different Expressed miRNAs
Two µg of total RNA from each piglet were polyadenylated by poly(A) polymerase (PAP) at 37°C for 1 h in a 20 µL reaction mixture using Poly(A) Tailing Kit (AM1350, Ambion, USA) according to the manufacturer’s instructions. Treated RNA was then dissolved and reverse-transcribed using poly (T) adapter and M-MLV (Promega, USA). qPCR for the 18 miRNAs was performed using SYBR Green Real-time PCR Master Mix (TaKaRa, Japan) in Mx3000P (Stratagene, USA) with a miRNA specific forward primer and a universal reverse primer complementary to part of the poly(T) adapter sequence. U6 was chosen as an internal control to normalize the technical variations. The sequences for all the primers and the poly (T) adapter are listed in Table S4. The method of 2−ΔΔCT was used to analyze the real-time PCR data expressed as the fold change relative to the average value of the LW piglets [43]. The differences between the two groups were analyzed by the student t-test for independent samples with the SPSS 13.0 for Windows.
