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Quantitative real-time PCR (QPCR) has become one of the most powerful quantification methods and a favorite tool in mRNA expression analysis and virus loading [12]. Because of its extreme sensitivity and accuracy, QPCR data analysis depends on a reliable reference gene to normalize for sample-to-sample and run-to-run variation, as variations arise from differences in nucleic acid integrity, the efficiency of the reverse transcription, and the amount of sample loaded [13, 23]. However, a number of studies have suggested that the most stable reference genes may vary between cell types, tissues, and even different physiological and disease states [3, 4, 14, 19, 20, 22]. Similarly, the ideal stable reference genes can also vary between different cell types infected with different viruses [14, 23]. So, selection of a stable reference gene is critical for reliable performance of QPCR experiments.
The highly pathogenic avian influenza caused by H5N1 has had devastating consequences for poultry production [1, 5, 24], and the virus has resulted in numerous infections in humans [25], making understanding of H5N1 viruses increasingly critical for public health. Quantitative analysis of H5N1 AIV and host mRNA levels is an important tool for the study of host-virus interaction. CEFs, the most commonly used cells in the study of host-avian virusinteraction [9, 17], are the most popular cells used in the study of H5N1 AIV [15]. However, no determination of the ideal reference genes for QPCR in these cells has yet been carried out in the context of H5N1 AIV infection. In this study, the expression stabilities of 11 housekeeping genes commonly used in mammals were compared, in order to select a stable reference gene in normal CEFs and H5N1 AIV infected CEFs.
The eleven housekeeping genes examined were as follows: albumin (ALB), beta-2-microglobulin (B2M), ribosomal protein L4 (RPL4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L30 (RPL30), hypoxanthine phosphoribosyltransferase 1 (HPRT1), succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA), TATA box binding protein (TBP), tubulin, beta (TUBB), tyrosine 3-monooxygenase, tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and the β-actin gene (ACTB).
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H5N1 AIV virus isolate (CHSD003), identified and purified by the China Animal Health and Epidemiology Center, was propagated in SPF chicken embryos. The TCID50 of the virus was determined in CEFs and calculated to be 107.67/0.1 mL according to Reed-Muench [27].
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CEFs were cultured from 10-day-old SPF chicken embryos according to standard procedures [27]. Briefly, 1× 107 cells were added per well in a 24-well culture plate. Monolayer cultures of CEFs were infected with 100 TCID50 H5N1 AIV. Cells were harvested at 3h, 12h, 24h and 30h post-infection with RNAiso Reagent Trizol (TakaRa). Mock-infected CEFs were cultured and harvested in the same way. Five parallel samples were taken for each time point.
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Total RNA from each sample was extracted by RNAiso Reagent (TaKaRa) and prepared using an RNase-free DNase kit (TakaRa) according to the manufacturer's recommendations. RNA was quantified by Cary50Probe (Bio-Rad) to find the OD260/OD280 value for each sample. The OD260/OD280 values of all the RNA samples were between 1.8 and 2.0, and intact rRNA subunits of 28S, 18S and 5S were observed on gel electrophoresis, indicating that all the RNA samples used in this study were of good quality.
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cDNA was synthesized using the RT Reagents (BioBRK) with random primer according to the manufacturer's recommendations. cDNA synthesis was performed in a PCR instrument(Bio-rad) using 1μg of RNA, at 30℃ for 10min, 42℃ for 20min, 99℃ for 5min, and finished at 4℃. Then the cDNA were treated with RNaseH in order to ensure the cDNA was without RNA. Finally, cDNA was saved at -20℃ for further testing.
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All primers (shown in Table 1) of the eleven housekeeping genes were designed by the Primer 5.0 software package and checked by the oligo 6.0 software tool. The positive pMD-Recombined Plasmids inserted with the purified PCR products of housekeeping genes were sequenced (Unbiotech) to verify the validity of PCR amplification.
Table 1. nformation for primers of real-time PCR
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The annealing temperature and primer concentration were optimized for all 11 housekeeping genes. Real-time PCR was performed in an ABI 7300 Real-time PCR System (Applied Biosystems) in 96-well microtiter plates using a final volume of 20 μL. Reactions were performed in triplicate for each sample, and the mean value for each sample was calculated. Electrophoresis analysis of all the amplified products from real-time PCR showed single bands with the expected sizes, and no primer dimer was observed. The dissociation plots provided by the ABI 7300 also showed a single peak for each reaction.
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Standard curves were generated using copy number vs. threshold cycle (Ct). The linear correlation coefficients (R2) of all eleven housekeeping genes were between 0.986 and 0.998. Based on the slopes of the standard curves, amplification efficiencies were between 95.66% and 109.95%, as derived from the formula E = 101/-slope -1. The Ct values of the standard curves of all the housekeeping genes had wide ranges.
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The expression levels of eleven housekeeping genes were measured by calculating the Ct of each by real-time PCR, and the expression stabilities were evaluated by the GeNorm tool [21], which determined the most stable housekeeping genes from a set of tested genes in a given cDNA sample panel. Relative expression levels of each housekeeping gene were the average value of all the samples of each gene at all the time points (3, 12, 24 and 30 hours post-infection) obtained by using the 2-△△Ct calculation method. [6, 21].
Virus propagation and the detection of TCID50
Viral infection of CEFs
Extraction of RNA
cDNA synthesis
Primer design and sequencing of PCR products
Real-time PCR of housekeeping genes
Standard curve of real-time PCR
Determination of stability and expression levels of housekeeping genes
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The average expression stability M values of 11 housekeeping genes are shown in Table 2. The expression stability ranking from the most stable to the least stable was: GAPDH, HPRT1, RPL4, RPL30, ACTB, YWHAZ, B2M, ALB, TBP, SDHA and TUBB. The ranking of the relative expression levels (from high to low) was: ACTB, RPL4, GAPDH, YWHAZ, HRRT1, TBP, TUBB, RPL30, SDHA, ALB and B2M. Based on both the best expression stability and highest abundance gene transcripts in the normal CEFs, GAPDH and HRRT1 were the two best reference genes.
Table 2. The average expression stability M value and relative expression levels of housekeeping genes in infected H5N1 AIV CEF and normal CEF
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The average expression stability M value of 11 housekeeping genes were shown in Table 2. The ranking of the expression stability (from the most stable to the least stable) was: ACTB, RPL4, YWHAZ, SDHA, GAPDH, B2M, TBP, ALB, HRRT1, RPL30 and TUBB. The ranking of the relative expression levels (from high to low) was: ACTB, RPL4, GAPDH, YHWAZ, HRRT1, TUBB, RPL30, TBP, SDHA, ALB and B2M. Based on both the best expression stability and high abundance gene transcripts, ACTB and RPL4 were the two best ideal reference genes in the infected CEFs.
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The average expression stability M values of 11 housekeeping genes (both in normal CEFs and CEFs infected with H5N1 AIV) were evaluated with the GeNorm tool and shown in Table 2. The ranking of the expression stability (from the most stable to the least stable) was: RPL4 and YWHAZ, ACTB, GAPDH, SDHA, B2M, TBP, HPRT1, ALB, RPL30 and TUBB. Based on both the expression stability and expression levels, RPL4 and YWHAZ were determined to be the two best reference genes for normalization of quantitative real-time PCR analysis of mRNA levels in host genes responses to H5N1 AIV.