Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells

<div><p>Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.</p></div

Comparison of mRNA quantitation by NanoString between unfixed and fixed cells.

<p>Eighteen human reference gene mRNAs were quantified using NanoString technology in unfixed and fixed samples of K562 (A) and CEMx174 (C) cell lines and human PBMCs (E). Correlation analyses of gene expression levels between unfixed and fixed samples of all cell types are shown (B, D, F). Each point represents one gene and points with zero value were not depicted on graph. Pearson correlation analysis was performed for all data sets. All values shown represent the average value of three biological replicates for unfixed and fixed.</p

Comparison of SIV DNA quantitation by qPCR between unfixed and fixed cells.

<p>SIV (A) and IFNβ (B) DNA copies per total DNA input were measured by qPCR in unfixed and fixed SIV-infected, pigtailed macaque PBMCs from three animals. Calculation of SIV copies per 100,000 cells for each sample was performed using respective IFNβ levels to normalize (C). Each point represents the average value of three technical replicates for one animal. Data were analyzed by Wilcoxon matched-pairs rank test.</p

Analysis of RNA integrity in unfixed and fixed cells.

<p>RNA integrity analysis of RNA isolated from unfixed and fixed samples was performed using Agilent 2100 Bioanalyzer. Representative electropherograms for unfixed (A, C, E) and fixed (B, D, F) samples of each cell type, K562 (A, B), CEMx174 (C, D), human PBMCs (E, F), are shown. The RNA Integrity Number (RIN) is shown for each sample.</p

Comparison of mRNA quantitation by RT-qPCR between unfixed and fixed cells.

<p>18S (A), GAPDH (B), TNFα (C) and MxA (D) mRNA quantitation by RT-qPCR were compared between unfixed and fixed samples of two cell lines, K562 and CEMx174, and human PBMCs. Individual points represent the difference of Ct values between unfixed and fixed samples for each gene obtained from one experiment (ΔCt = Fixed Ct – Unfixed Ct). Three biological replicates are shown for each sample. <i>p</i> values were calculated by a one-sample, two-tailed t test comparing the average ΔCt to a theoretical mean set to zero. Statistically significant <i>p</i> values are shown.</p

siRNA silencing of PREP1 and PBX2 in transfected U87-MG cells differentially affects promoter activity of the -2578 A and G alleles.

<p><b>A,B.</b> Luciferase expression in U87-MG cells transfected with either the -2578 A pGL4.11 plasmid or the -2578 G pGL4.11 plasmid along with indicated siRNAs. <b>C.</b> Luciferase expression in U87-MG cells transfected with either the -2578 A pGL4.11 plasmid or the -2578 A<i>m</i> pGL4.11 plasmid. <b>D.</b> Luciferase expression in U87-MG cells transfected with either the -2578 G pGL4.11 plasmid or the -2578 G<i>m</i> pGL4.11 plasmid. All results were normalized to Renilla luciferase values. Results are expressed as the mean ± S.E. of three independent experiments carried out in duplicate. Differences between multiple groups were tested using ANOVA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022052#pone-0022052-g003" target="_blank">Figure 3A</a>, P = 0.0495 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022052#pone-0022052-g003" target="_blank">Figure 3B</a>, P<.0001). Subsequent pair wise comparisons of groups with each treatment against the control group utilized the Bonferroni correction. Student's two-sided <i>t</i> tests were used for 3C and 3D.</p

Distinct TALE site binding preferences for PREP1, PBX2, HoxA9 and IRF1 in the CCL2 promoter.

<p>ChIP results from U87-MG cells transfected with appropriate plasmids (as described above) and activated with IL-1β for 3 hours. <b>A.</b> Western blot results from U87-MG cells transfected with indicated siRNAs. GAPDH represents loading control. <b>B,C.</b> ChIP results from U87-MG cells co-transfected with the -2578 G pGL4.11 plasmid and indicated siRNA. Graphics show percent of total DNA immunoprecipitated by each indicated antibody. IgG represents the negative control. Results are expressed as the mean ± S.E. of three independent experiments carried out in duplicate. Differences between multiple groups were tested using ANOVA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022052#pone-0022052-g001" target="_blank">Figure 1B</a>, P = 0.0115 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022052#pone-0022052-g001" target="_blank">Figure 1C</a>, P = 0.0014). Subsequent pair wise comparisons of groups with each treatment against the control group utilized the Bonferroni correction.</p

Distinct TALE site binding preferences for PREP1, PBX2, HoxA9 and IRF1 in the CCL2 promoter with and without TALE site repeats.

<p><b>A.</b> Sequence of double stranded CCL2 promoter DNA representing the distal regulatory region containing the two TALE binding sites (in red) with the A-2578G polymorphism within parentheses and the incorporated mutation within the upstream TALE site (denoted by *<i>m</i>). The CCL2 ISRE sequence is within the black box. Graphics show the percent of total DNA immunoprecipitated from U87-MG cells by each indicated antibody. <b>B-I.</b> U87-MG cells were transfected with either the -2578 A pGL4.11 plasmid or the -2578 A<i>m</i> pGL4.11 plasmid. <b>F-I.</b> U87-MG cells were transfected with either the -2578 G pGL4.11 plasmid or the -2578 G<i>m</i> pGL4.11 plasmid. Percent input was calculated by 100×2∧(Ct_input – Ct_enriched). Input was determined from 1% of the cell lysate and results are expressed as the mean ± S.E. of three independent experiments carried out in duplicate.</p

Sequence analysis of wild-type (WT) and variant LTRs detected in the spleen and brain.

<p>(A) Consensus sequence of variant LTR genotype groups were aligned to reference, wild-type (SIV/17E-Fr mLTR: −236 to −154 bp) sequence. Dashes (-) indicate conserved nucleotides; insertions and deletions are denoted by (:), horizontal rectangles highlight the JC1 and DS1 C/EBP sites. Vertical rectangles highlight +123 bp A/G and +146C/A substitutions within the DS1 C/EBP site. (*) 22nt variant found in 1 of 180 cloned sequences from the SIV/DeltaB670 swarm virus stock. (B) Frequency of LTR genotypes in spleen RNA from the indicated animals euthanized at 10, 21, 42, 48 and 84 days p.i. is depicted. Bars with gray/black background indicate the presence of DS1C/A in the LTRs sequenced. Bars with white background indicate absence of DS1C/A substitutions. (C) Genotype frequency of LTRs sequenced from brain RNA/DNA homogenates from each individual animal, as indicated. (¥) RNA derived LTR sequencing results for one 21 days p.i. animal, PBc2, are shown next to DNA derived LTR sequencing results from the same animal. (†) Indicates variant genotypes that are non-wild-type but lack the DS1C/A substitution.</p

Early Emergence and Selection of a SIV-LTR C/EBP Site Variant in SIV-Infected Macaques That Increases Virus Infectivity

<div><p>CCAAT/enhancer binding protein (C/EBP)β, and C/EBP binding sites in the HIV/SIV- long terminal repeat (LTR) are crucial for regulating transcription and for IFNβ-mediated suppression of virus replication in macrophages, the predominant source of productive virus replication in the brain. We investigated sequence variation within the SIV-LTR C/EBP sites that may be under selective pressure <em>in vivo</em> and therefore associated with disease progression. Using the SIV-macaque model, we examined viral LTR sequences derived from the spleen, a site of macrophage and lymphocyte infection, and the brain from macaques euthanized at 10, 21, 42, 48 and 84 days postinoculation (p.i.). A dominant variant, DS1C/A, containing an adenine-to-guanine substitution and a linked cytosine-to-adenine substitution in the downstream (DS1) C/EBP site, was detected in the spleen at 10 days p.i. The DS1C/A genotype was not detected in the brain until 42 days p.i., after which it was the predominant replicating genotype in both brain and spleen. Functional characterization of the DS1C/A containing SIV showed increased infectivity with or without IFNβ treatment over the wild-type virus, SIV/17E-Fr. The DS1C/A C/EBP site had higher affinity for both protein isoforms of C/EBPβ compared to the wild-type DS1 C/EBP site. Cytokine expression in spleen compared to brain implicated IFNβ and IL-6 responses as part of the selective pressures contributing to emergence of the DS1C/A genotype <em>in vivo</em>. These studies demonstrate selective replication of virus containing the DS1C/A genotype that either emerges very early in spleen and spreads to the brain, or evolves independently in the brain when IFNβ and IL-6 levels are similar to that found in spleen earlier in infection.</p> </div