tissue: superior vena cava, adipose tissue, lung, spleen, stomach, liver, intestine, kidney, descending aorta, left atrium, left ventricle, skeletal muscle, pulmonary aorta, skin, tongue, ascending aorta, arterial white cells blood, venal white cells blood, coronary valve, lymph node age: 12 month old breed: vietnamite
Treatment protocol
After the sedation, pigs were sacrificed and portions of the described organs were took and maintained in the RNALater solution (Ambion), excluding blood that was collected in the BD Vacutainer CPT cell preparation tubes, and processed according to manufacturer's directions to recover white blood cells.
Growth protocol
Pigs were maintained at normal food regime
Extracted molecule
total RNA
Extraction protocol
Total RNA and miRNAs were extracted independently from each tissue sample by TRIzol reagent (Invitrogen) in association with PureLink miRNA isolation kit (Invitrogen). Briefly, approximately 200 mg of tissue was homogenized in 3.5 ml of TRIzol reagent (Invitrogen) using a tissue homogenizer (IKA Werke). After chloroform addition and centrifugation colorless upper aqueous phase containing RNAs was added with 96-100% ethanol to obtain a final concentration of 35% of ethanol and charged to PureLink membrane (Invitrogen) to separate total RNA and small RNAs from the same sample following the manufacturer manual. Total RNA and small RNAs were quantized using Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific). Samples derived from the same tissues were pooled adding the same quantity from the three pigs used. “Pool tissues” were performed with 700 ng of small RNAs from each pool sample. Quality of small RNA of pooled samples was tested on Agilent Bioanalizer 2100 using the RNA small LabChip.
Label
Cy3
Label protocol
The small RNA was not labelled when it was used to probe microarray. RNA-primed Array-based Klenow Extension (RAKE) is based on the ability of a RNA molecule to function as a primer for Klenow polymerase extension when fully base-paired with a microarray probe. As the exact 3’ end of the miRNA should be known for successful extension, and computational predictions are not optimal for predicting the correct start and end of the mature miRNA, we designed a tiling path of 16 probes complementary to the predicted pre-miRNAs responsive for the 3’ end of the miRNA. The protocol for the identification of the 5’ end of miRNAs was the same used for the identification of the 3’ end, but the 12K microarray was probed with the retrotranscribed miRNAs (see Figure). A poly(A) tail was added to small RNAs from the same pool used for the identification of the 3’ end of miRNAs. 1 μg of small RNAs was added to the polyadenilation mix (E-PAP Buffer 1X; MnCl2 2.5 mM; ATP 1mM; E-Polyadenylase Polymerase Invitrogen 0.08 U/μl in 100 μl of reaction) incubated for 1 hour at 37° C than precipitated using Na-Acetate and 4 volumes of ethanol. Tailed small RNAs were retrotranscribed with Superscript III (Invitrogen) enzyme that does not have terminal-transferase activity. Retrotranscription was performed incubating the mix (First strand Buffer 1X; DTT 1 mM; dNTPs 0.5 mM; Superscript III enzyme Invitrogen 8 U/μl in 50 μl of volume reaction) for 1 hour at 42° C.
Hybridization protocol
Microarrays were probed with 1 ug of the retrotranscribed small RNA pool for 20 hours at 37° C in a static hybridization oven (SSPE 6X; BSA 8 mg/ml; 1 ug of small RNAs and spike-in). First, microarrays were pre-hybridized for 2 hours at 37° C with the following pre-hybridization water solution: SSPE 6X and BSA 8 mg/ml. After the retrotranscribed small RNAs hybridization, microarrays were washed with the following stringent washing solutions: 1 minute at room temperature with 6x SSPET (SSPE added with 0.05% of Tween-20); 1 minute at room temperature with 3x SSPET; 1 minute at room temperature with PBS 2X; 1 minute at room temperature with Buffer 2, 1X (the buffer for the klenow enzyme). After the washing step it was performed the RAKE reaction at 36.5° C incubating the microarray for 1 hour and 30 minutes with the following solution: Buffer 2 1X; Biotin-14-dATP (Invitrogen) 16 μM; Klenow Fragment (3´→5´ exo–) (NEB) 0.25 U/μl. Than microarray was washed two times with Buffer 2 1X and than incubated with the biotin blocking solution (PBS 2X; Tween-20 0.1% and BSA 10 mg/ml) for one hour at room temperature. Extended miRNAs (primers) were labeled incubating the microarray with the Dye labeling solution (PBS 2X; Tween-20 0.1% ; BSA 10 mg/ml and 1.6 ng of Cy3-streptavidin; Amersham) for one hour at room temperature. Microarray was rinsed with PBST (PBS 2X added with Tween-20 0.1%) for one minute at room temperature and with PBS 2X for one minute at room temperature (see Figure).
Scan protocol
Microarrays were scanned with the VersArray scanner (Biorad) (3 μm resolution).
Description
Tissue was extracted from three different pig (12 month old) and small RNA was pooled. Retrotranscribed small RNA RAKE technology. Extended primer (hybridized miRNA) incorporate dATP biotinilated than labelled with Cy3-streptavidin RNA-primed Array-based Klenow Extension (RAKE) is based on the ability of a RNA molecule to function as a primer for Klenow polymerase extension when fully base-paired with a microarray probe. As the exact 3’ end of the miRNA should be known for successful extension, and computational predictions are not optimal for predicting the correct start and end of the mature miRNA, we designed a tiling path of 16 probes complementary to the predicted pre-miRNAs responding for the presence of a 3’ end of the miRNA (see Figure).
Data processing
The RAKE experimental setup associates fluorescence levels to 16 tailed probes corresponding to the 3' ends of each retrotranscribed miRNA (see Figure). To identify the most probable endpoints, we then need to analyze these values looking for spots with a fluorescence level significantly higher from the rest of the group. We implemented this search with a bootstrap approach. Given a group of 16 measures, we extract in turn one of them and we build ten thousand simulated groups by sampling the remaining 15 values with replacement. The number of samples with an average fluorescence level lower than the value of the observed group, divided by ten thousand, is taken as an estimate of the probability of having picked a true endpoint for the miRNA. For the rest of the analysis, we keep only the results with a probability higher than 0.65 and for a minor group . 5’ end had a probability higher than 0.58. Data for the identification of the 3' end of the miRNAs (in this case 3' end of the retrotranscribed miRNA correspond to the 5' end of the mature miRNA) did not need to be normalized because we did not compared different experiments, but we extracted from each experiments peaks corresponding to miRNA end.
Discovering, evolution, biogenesis, expression and target prediction of porcine micro-RNAs: new regulatory gene expression network in different tissues.
