Quantitative polymerase chain reaction was used for quantitative analysis of gene expression of investigated parameters during this study. Each inspected parameter was chosen due to a potential direct or indirect impact on the transport physiology of the enterocytes of the small intestines. Since the present study results from a cooperation project with the Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Foundation, Buenteweg 17, 30559 Hannover, Germany, the qPCR was conducted by Sarina Köhler from the aforementioned institute.
2.5.1. qPCR - Design of primer and probe
To gain reliable results, suitable primer and probes for the specific target genes need to be designed for which the GenBank (NCBI) database was used. This provided the nucleotide sequences of selected genes derived from the Sus scrofa genome. The Clone Manager Software (Version Professional 9, Sci Ed Software, Westminster, CO, USA) was used to align multiple sequences and based on those alignments, gene-specific primers and TaqMan™ probes were designed with primer Allele-ID software (PREMIER Biosoft, San Francisco, CA, USA). The primer pairs and TaqMan™ probes used in these experimental runs including the accession numbers can be found in Table 2. Based on GenBank accession numbers, primers were ordered from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) and primer sequences are available on request. Due to the fluorescent labelling (Life Technologies GmbH, Darmstadt, Germany) of the probes at the 3’end with VIC® or FAM™, the simultaneous detection of two genes was possible. A non-fluorescent quencher was attached to the 5’end.
For the preparation of plasmid standard, available mRNA sequences on GenBank® (NCBI) were aligned for each gene and Primer Blast (NCBI) was used to choose the best matching sequences for primer pair design.
Gene Forward primer (5’-3’)
Gene Reverse
primer (5’-3’) TaqMan™ MGB probe Amplicon
length (bp) Accession number
Table 2. TaqMan™ MGB probes for quantitative real-time PCR and Primer pairs including accession numbers
2.5.2. qPCR – Extraction of mRNA and synthesis of cDNA
For mRNA extraction 30-40µg mucosa of each segment from each pig was homogenised in lysis solution (GenElute™ Direct mRNA Miniprep Kit, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) using the Precellys metal kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) at 6400 rpm for 60 seconds in the Precellys® 24 tissue homogenizer (PEQLAB Biotechnologie GmbH, Erlangen, Germany).
Subsequently, mRNA was isolated with the GenElute™ Direct mRNA Miniprep Kit (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Quality and quantity were determined based on the use of the NanoDrop™ 1000 spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Overnight, ethanol precipitation was performed, followed by mixing 100 µL of the sample with 5 µL 3M sodium acetate buffer (pH 5.2), 300 µL ice-cold ethanol (95%) and 20 ng glycogen. At 16,000 g and 4 °C, the samples were centrifuged for 30 min. On the following morning, supernatant was removed and replaced with 500 µL of cold ethanol (70%), centrifuged for 10 min at 16,000 g and again ethanol was removed. The formed pellet was dried and afterwards resuspended in 21 µL of RNAse-free water. The NanoDrop™ 1000 spectrophotometer was utilised to determine quality and quantity for a second time.
Using 20 μL precipated mRNA, the RNA to cDNA EcoDry™ Premix (OligodT) stripes (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) were used to convert mRNA to cDNA. For a third
time, quality and quantity were determined and cDNA was diluted 1:10 in RNAse-free water afterwards. Until use, the cDNA was kept at -80 °C.
2.5.3. qPCR – Preparation of plasmid standard
For quantification of specific amplification efficiencies of the target and reference (housekeeping) genes, plasmid standard dilution series were produced, these dilution series being included in each run. For each gene aligned mRNA sequences available on GenBank® (NCBI) were used.
For amplification, a total volume of 50 µL per reaction for the first protocol of TBP, GLUT2, SGLT1, IL-13 was used. For the performance of the protocol, 38 µL distilled water, 5 µL 10x Taq buffer, 1 µL 10 mM desoxynucleotide triphosphates, 2µL of 10 µM forward and reverse primer each and 1 µL PerfectTaq DNA polymerase (5 U/reaction. 5 Prime GmbH, Hilden, Germany) were mixed.
Furthermore, Qiagen Multiplex Mix (Qiagen GmbH, Hilden, Germany) was used for PPIA, GLUT1, Hif-1α, IL-4 and PepT1 amplification in a 50 µL reaction. In both protocols, 1 μL cDNA (200 ng/µL) derived from mucosa of the jejunum of a pig in the control group was added as a template.
For the 5Prime Perfect Taq PCR, the thermocycling protocol was conducted as follows: it was started at 95 °C for 3 min, denaturation followed for 40 cycles at 95 °C and 30 sec. Varying respective temperatures, primer annealing was performed at 59 °C for IL 13, 60 °C for SGLT1 and for TBP and STAT6 at 61 °C for 30 sec, respectively. Afterwards, primers were extended at 72 °C for 30 sec and the protocol ended by elongation for 10 min at 72 °C.
For the Qiagen Multiplex (Qiagen GmbH, Hilden, Germany), the thermoprofile was started at 95 °C for 15 min, followed by 40 cycles for 30 sec at 94 °C. The annealing followed for 90 sec at 59 °C for GLUT1, 60 °C for PPIA and IL 4 and at 61 °C for HIF1A and PepT1, respectively. This was followed by primer extension for 30 sec at 72 °C and final elongation at 72 °C for 10 min.
Products resulting from amplification (4 µL) were ligated to TOPO™ TA vectors and then transformed One Shot™ TOP10 Chemically Competent E. coli (both Invitrogen™, Thermo Fisher Scientific GmbH, Schwerte, Germany). After sequencing, the plasmid-DNA was purified from E. coli cells. Subsequently, linearisation and dephosphorylation in accordance with Laabs et al. followed (Laabs et al. 2012). The usage of the NanoDrop™ 1000 spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany) at an absorption spectre of 260 nm allowed aliquoting of 100 to 106 copies per µL. Until single use, aliquots were stored at -20 °C.
2.5.4. qPCR – Performance
Of the two appropriate genes, 12.50 μL ABsolute™ Blue QPCR Low ROX Mix (Thermo Fisher Scientific GmbH, Schwerte, Germany), 0.15 μL forward and 0.15 μl reverse primer (50 μM) as well as 0.06 μL probe (10 μM) for each duplex qPCR reaction were mixed. The following duplex pairs were used: PPIA together with TBP, GLUT1 together with GLUT2, SGLT1 together with IL-4, Hif-1α together with IL13 and PepT1 together with STAT6, respectively. Of the porcine intestinal mucosa, 2 μL of the first-strand cDNA preparations and 1 μL of each ten-fold diluted serial plasmid standard were used as template.
The thermoprofile consisted of 15 min at 95 °C, followed by 40 cycles of 95 °C for 20 sec, 52 °C for 20 sec and 72 °C for 30 sec. All reactions were run in duplicate and the reference genes TBP and PPIA were included on each 96-well microplate. Each plate run was repeated once. The mean gene-specific amplification efficiency, correlation coefficient (R2), slope value and y-intercept in duplex reactions were analysed (see Tab. 3). qBase+ software (Version 3.2, Biogazelle, Ghent, Belgium) was used for normalisation of average baseline-corrected reporter signals of duplicates and technical replicates, correction of run-specific amplification efficiencies, inter-run calibration and normalisation against reference genes.