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This work represents the first genome-wide analysis of histone acetylation and its …


Biology Articles » Parasitology » Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica » Methods

Methods
- Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica

Entamoeba strains and culture methods

The strains used in this study were E. histolytica HM-1:IMSS and E. histolytica 200:NIH both of which were grown axenically in TYI-S-33 medium under standard culture conditions at 36.5°C [35]. Additionally, both strains were grown in TYI-S-33 medium in the absence of glucose (LG medium), in TYI-S-33 medium plus SCFA (70 mM sodium acetate, 20 mM sodium propionate and 10 mM sodium butyrate) (SCFA medium) [23], and in TYI-S-33 medium plus TSA (150 nM or 300 nM) (TSA medium). Genotypes of the E. histolytica strains (HM-1:IMSS and 200:NIH) were confirmed by PCR and RFLP based on previously published methods [36,37].

RNA isolation and microarray hybridization

Total RNA was isolated using Trizol reagent (Invitrogen) using the manufacturer's protocol and purified using a Qiagen RNeasy kit before being used for microarray analysis [14]. Samples were processed for microarray hybridization by the Stanford University Protein and Nucleic Acids facility [38] using standard protocols. For each sample the RNA quality was checked using an Agilent BioAnalyzer QC and 4 μg subjected to the standard labeling and hybridization method [39]. E. histolytica 200:NIH parasites were grown in SCFA for 16 hours, harvested and RNA extracted for microarray experiments. E. histolytica 200:NIH parasites were grown in TSA (150 nM or 300 nM) for 16 or 72 hours (150 nM), harvested and RNA extracted for microarray experiments.

Labeled samples were hybridized to a custom generated Affymetrix platform full genome microarray (E_his-1a520285F), which has been previously described [27]. This array has 7,712 unique probe sets, which represent 9,435 open reading frames. Due to the highly repetitive nature of the E. histolytica genome, some of the probe sets are predicted to cross-hybridize with other sequences. Probe sets that represent a single gene and do not cross hybridize are labeled as (_at). Probe sets in which at least one probe may cross-hybridize with another gene(s) are labeled as (_x_at). In situations where all the probes for a given gene cross-hybridize with another gene(s), the probe sets is labeled as (_s_at) and additional genes that cross-hybridize with this probe set are listed in Additional File 3. This array also contains probes for intergenic non-coding regions, however, these probe sets were excluded from all analysis. After hybridization, arrays were scanned and probe intensities calculation using Affymetrix GCOS software [40].

Microarray data normalization and analysis

Normalized expression values for each probe set were obtained from raw probe intensities in R 2.2.0 downloaded from the BioConductor project [41], using robust multi-array averaging with correction for oligo sequence (gcRMA) [42]. To identify differentially expressed genes, we used local pooled error testing [43] along with Benjamini-Hochberg multiple test correction [44]. In addition, fold-change was calculated in Genespring GX [45]. A minimum of three arrays from each condition were used for analysis of SCFA or TSA effects. Correlation coefficients were calculated in Genespring using standard correlation. Probe sets were considered differentially expressed between two conditions if they had at least a 2-fold change and were significant with a false discovery rate (FDR) of < 0.05, and were identified as "present" in at least one array. Datasets of transcriptional profiles from E. histolytica HM-1:IMSS, E. histolytica Rahman, parasites from an in vivo model of colitis, encystation, and 5-AzaC treatment were obtained from previously published data [14,16,27].

Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)

E. histolytica 200:NIH trophozoites grown in TYI-S-33 LG medium were transferred in mid log phase (3 × 105 per ml) into TYI-S-33 LG or TYI-S-33 LG/150nM TSA and incubated for 16 hrs. Total RNA was isolated with RNAzol, and 2ug RNA was treated with DNAase I for 5 minutes at 37°C. cDNA was synthesized with oligo-dT and Superscript III reverse transcriptase (Invitrogen) at 50°C for 2 hr. Ten-fold dilutions of cDNA were used as template for 30 cycles of PCR amplification with gene-specific primers. PCR products were fractionated on 1.5% agarose gels, stained with ethidium bromide, and photographed with a GE/Amersham ImageQuant ECL recorder. Primers used in the study are:

135.m00113 Sense (5'-CCGAATCTGCATTTCCAACT-3') and

135.m00113 Antisense (5'-CAATCCCTCCTCCAAGTGAA-3');

135.m00113 Sense (5'-TCTACTTGGAGGAGGGATTC-3') and

135.m00113 Antisense (5'-AATGAATTTGCATTGCATGG-3');

14.m00310 Sense (5'-GCCAGTTTCATTCCATGGTT-3') and

14.m00310 Antisense (5'-TCAGGACCACCAACATTTGA-3');

337.m00049 Sense (5'-TCAATGAATTGGTCGTTTGC-3') and

337.m00049 Antisense (5'-TCGTTTTGGTGTGAAATGTTG-3');

146.m00117 Sense (5'-CCCCATCCAAAATTGAACAG-3') and

146.m00117 Antisense (5'-GGATGGGGATTAGAAACCAAA-3');

223.m00071 Sense (5'-CCTAAACTTCAGCAAGTTCATTCA-3') and

223.m00071 Antisense (5'-GAAAGAAGTTGAGCCCAAAGCA-3');

1.m00712 Sense (5'-AACAATTGGTCAATGCTTCTCA-3') and

1.m00712 Antisense (5'-TCCCAAATGAACGAATAGGC-3');

223.m00075 Sense (5'-TGCAAAAATTAATAACCTTCTTCG-3') and

223.m00075 Antisense (5'-TCCACCAACAAAACCTGAAA-3');

77.m00173 Sense (5'-CAACATCTATTGGAAAAAGACCA-3') and

77.m00173 Antisense (5'-TGGAGATAACTCCTTCTCCATCA-3');

340.m00050 Sense (5'-CATCGAATATGATATTACATCAAATG-3') and

340.m00050 Antisense (5'-TTTATTGGAATTGGGTCAATAGCATTC-3');

247.m00075 Sense (5'-TGCAAAGTCCATTTCCAACA-3') and

247.m00075 Antisense (5'-TTTCAGGAGAAAAAGTGGCTTC-3');

7.m00480 Sense (5'-TGATTGCAAAAGATTCAGAAACA-3') and

7.m00480 Antisense (5'-ACTTGACCCAAAGTCATCACG-3');

13.m00291 Sense (5'-TGCTCAATGGCATCAATGTT-3') and

13.m00291 Antisense (5-'GCTTCCATTTGGGACGTAGA-3');

ssRNA Sense: (5'-ACGAACGAGACTGAAACCTAT-3') and

ssRNA Antisense: (5'-TGTTACGACTTCTCCTTCCTC-3').


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