Abstract
Natural antisense transcripts (NATs) have been detected in many organisms and shown to regulate gene expression. Similarly, NATs have also been observed in malaria parasites with most studies focused on Plasmodium falciparum. There were no reports on the presence of NATs in Plasmodium vivax, which has also been shown to cause severe malaria like P. falciparum, until a recent study published by us. To identify in vivo prevalence of antisense transcripts in P. vivax clinical isolates, we performed whole genome expression profiling using a custom designed strand-specific microarray that contains probes for both sense and antisense strands. Here we describe the experimental methods and analysis of the microarray data available in Gene Expression Omnibus (GEO) under GSE45165. Our data provides a resource for exploring the presence of antisense transcripts in P. vivax isolated from patients showing varying clinical symptoms. Related information about the description and interpretation of the data can be found in a recent publication by Boopathi and colleagues in Infection, Genetics and Evolution 2013.
Keywords: Natural antisense transcripts, Strand specific microarray, Plasmodium vivax, Complicated malaria, Uncomplicated malaria
| Specifications | |
|---|---|
| Organism/cell line/tissue | Plasmodium vivax |
| Source | P. vivax parasites from patients' blood |
| Sequencer or array type | Agilent custom P. vivax 244K array. (AMADID: 019103) |
| Data format | Raw data: txt files, normalized data: SOFT, MINiMAL, TXT |
| Experimental factors | Complicated and uncomplicated P. vivax malaria |
| Experimental features | Identification of sense and antisense transcripts in P. vivax isolated from patients showing differing clinical symptoms. |
| Consent | The patient samples were collected on informed consent by the team of clinicians at S. P. Medical College, Bikaner, India according to hospital guidelines. |
| Sample source location | Bikaner, Rajasthan, India. |
Direct link to deposited data
Experimental design, materials and methods
Sample collection and preparation
Venous blood samples (~ 5 ml) were collected from 8 Plasmodium vivax infected adult patients on informed consent at S. P. Medical College, Bikaner, India. The patients were diagnosed as either complicated (n = 7) or uncomplicated malaria (n = 1). The infection with P. vivax was confirmed by detailed investigation of peripheral blood films (PBFs) and rapid diagnostic tests (RDTs) (Optimal test; Diamed AG, Cressier sur Morat, Switzerland, Falcivax test; Zephyr Biomedical System, Goa, India). All other laboratory investigations as described in [4] were also performed. Peripheral blood mononuclear cells (PBMCs) were separated from infected and non-infected erythrocytes using density gradient based separation (Histopaque 1077, Sigma Aldrich, USA) according to manufacturer's instructions. The infected and non-infected erythrocytes were washed with phosphate buffered saline (PBS) and lysed using Tri-Reagent (Sigma Aldrich, USA) and preserved immediately at − 80 °C. All the samples were then transported in cold chain to BITS, Pilani for further processing. The infection with only P. vivax was confirmed by 18S rRNA based multiplex PCR and 28S rRNA based nested PCR [6], [7].
Microarray hybridization and scanning
Total RNA and DNA was isolated from complicated (n = 7) and uncomplicated (n = 1) malaria blood samples, according to manufacturer's protocol (Tri-Reagent, Sigma Aldrich, USA). The quality of the isolated total RNA samples was analyzed by denaturing agarose gel and also by using RNA 6000 Nano Lab Chip on the 2100 Bioanalyzer (Agilent, Palo Alto, CA) following manufacturer's protocol. Quantity and purity of the total RNA was measured by the NanoDrop® ND-1000 UV–vis Spectrophotometer (Nanodrop technologies, Rockland, USA). The total RNA from 7 complicated malaria samples was pooled in an equimolar amount. Total RNA (500 ng) from each of the pooled complicated and uncomplicated malaria samples was amplified and labeled in the presence of Cyanine 5-CTP and Cyanine 3-CTP respectively using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) following manufacture's protocol. After labeling, the cRNA was cleaned and the quality was assessed for yields and specific activity. Then 1500 ng of each Cy3 and Cy5-labeled samples was mixed, fragmented and hybridized to the array at 65 °C for 16 h using Gene Expression Hybridization Kit (Agilent Technologies, Part Number 5188–5242). The hybridized slides were washed using Gene Expression wash buffers (Agilent Technologies, Part No: 5188–5327) and scanned using the Agilent Microarray Scanner (Agilent Technologies, Palo Alto, CA, G Model G2565BA) at 5 μm resolution. Image analysis was conducted using Agilent Feature extraction software (Agilent Technologies).
244K custom array designing
A custom genome-wide strand specific P. vivax 244 K microarray was designed on an Agilent platform using the RightDesign (Genotypic Technology, Bangalore, India) probe design workflow to choose the best probe(s) for a transcript by balancing several criteria: GC content, sequence complexity, cross hybridization potential and secondary structure. The array contains 232756 60-mer oligonucleotide probes (sense and antisense probes) representing P. vivax Sal-I transcript sequences from PlasmoDBv5.3 [1], [3], expressed sequence tags (ESTs) and whole genome shotgun assemblies of P. vivax from NCBI (2007) and apicoplast sequences of P. vivax [9] and Plasmodium falciparum [12]. Annotations of all the probes were again updated according to PlasmoDB v8.2 and NCBI database (2012). Information about the array was described in detail in [2]. Summary of the array details has been submitted and available under GEO accession number GPL16492. Information such as feature number, oligonucleotide probe ID and sequence, target gene identifier ID against which probe has been designed and the gene description is provided in the array details. Here, we also give additional information about the orientation of probe against the target gene in the array (Supplementary Table S1).
Data analysis
The raw signal intensities and background intensities were obtained for each channel and analyzed separately for uncomplicated (Green channel) and complicated (Red channel) malaria. Analyses of probe hybridization for the PlasmoDBv8.2 [1], [3] transcripts were discussed in the paper. A total of 5317 genes represented by both the sense and antisense probes were only considered for the analysis. Ratio was calculated for each probe by dividing the raw signal intensity and background signal intensity. Probes (sense or antisense) showing ≥ 2-fold the background intensity were filtered. Probe data were converted to gene based data. For genes with multiple probes, median of raw signal intensity and background intensity was considered. Genes with ≥ twice the median background signal intensity were included in further analysis. To these genes, we have applied a stringent filtering criterion by considering genes expressing only sense(S), antisense (AS) or both S and AS transcripts represented by at least 3 probes. We categorized genes based on the type of transcripts they expressed (1) genes with only S transcripts (2) genes with only AS transcripts and (3) genes with both S and AS transcripts. Classification of genes with S and AS transcripts in complicated P. vivax malaria (PVC) and uncomplicated P. vivax malaria (PVU) is shown in Table 1. We detected a total of 1348 Natural Antisense transcripts using strand-specific custom designed microarray. Detailed analysis of this study has been published [2].
Table 1.
Genes expressing sense and antisense transcripts in complicated and uncomplicated malaria.
| Genes expressing |
|||
|---|---|---|---|
| Only sense (S) transcripts | Only antisense (AS) transcripts | Both S and AS transcripts | |
| PVC | 959 | 106 | 942 |
| PVU | 782 | 117 | 934 |
Number of genes expressing AS transcripts in PVC is 1048.
Number of genes expressing AS transcripts in PVU is 1051.
Unique number of genes expressing AS transcripts in PVC & PVU is 1348.
Discussion
Here we describe information about microarray dataset obtained from our custom designed strand-specific genome-wide P. vivax array on an Agilent platform. The dataset comprises whole genome transcriptome profiling of P. vivax isolated from patients showing differing clinical symptoms. The dataset was analyzed in recently published study and is the first study to reveal the presence of NATs in P. vivax clinical isolates. Discovery of NATs in P. vivax and P. falciparum [2], [5], [8], [10], [11] suggests that they might play an important role in regulating gene expression. Results from this microarray dataset thus would greatly assist investigations of gene regulation in future.
The following are the supplementary data related to this article.
Details of probes in the strand specific 244 K P. vivax microarray.
This table lists details of the probes in the strand-specific 244 K P.vivax microarray, which were re-annotated against transcript sequences from PlasmoDBv8.2 [1], [3] and also against ESTs and genome sequences from NCBI (2012). Probes which could not be assigned to any of the sequences in the current database (PlasmoDBv8.2 & NCBI(2012)) during the re-annotation process has been removed. Probe list contains information about oligonucleotide probe ID, gene symbol, probe orientation, gene description, feature number, location of features and control type of features.
Conflict of interest
The authors declare that they have no conflicts of interest.
Acknowledgments
We thank all the patients and technical workers for their participation in support of this project. A.K.D., S.K.K. and D.K.K. acknowledges Department of Biotechnology (DBT), New Delhi, India for the financial support through the grant BT/PR7520/BRB/10/481/2006 and Birla Institute of Technology and Science, Pilani, India and S.P. Medical college, Bikaner, India for providing the required infrastructural facilities during this study. P.A.B. acknowledges Basic Scientific Research fellowship from University Grant Commission, New Delhi, India and Project Assistantship from Department of Biotechnology (DBT), New Delhi, India. A.K.S. acknowledges Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), New Delhi, India and Project Assistantship from Department of Biotechnology (DBT), New Delhi, India. We thank the PlasmoDB team for making available the genome of the Sal I strain. We also thank Genotypic Technology Pvt. Ltd., Bangalore, India for the microarray hybridization and preliminary data analysis service provided by them.
Contributor Information
P.A. Boopathi, Email: boopathiarunachalam@gmail.com.
Amit Kumar Subudhi, Email: amit4help@gmail.com.
Shilpi Garg, Email: shilpi_garg19@yahoo.co.in.
Sheetal Middha, Email: sheetumig21@gmail.com.
Jyoti Acharya, Email: jyotiacharya2@gmail.com.
Deepak Pakalapati, Email: deepakpchowdary@gmail.com.
Vishal Saxena, Email: vishalsaxena12@gmail.com.
Mohammed Aiyaz, Email: aiyaz@genotypic.co.in.
Bipin Chand, Email: bipin.c@genotypic.co.in.
Raja C. Mugasimangalam, Email: raja@genotypic.co.in.
Sanjay K. Kochar, Email: drskkochar@rediffmail.com.
Parmendra Sirohi, Email: drpsirohi@gmail.com.
Dhanpat K. Kochar, Email: drdkkochar@yahoo.com.
Ashis Das, Email: ashisd28@gmail.com, ashdas28@gmail.com, adas@pilani.bits-pilani.ac.in.
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Associated Data
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Supplementary Materials
Details of probes in the strand specific 244 K P. vivax microarray.
This table lists details of the probes in the strand-specific 244 K P.vivax microarray, which were re-annotated against transcript sequences from PlasmoDBv8.2 [1], [3] and also against ESTs and genome sequences from NCBI (2012). Probes which could not be assigned to any of the sequences in the current database (PlasmoDBv8.2 & NCBI(2012)) during the re-annotation process has been removed. Probe list contains information about oligonucleotide probe ID, gene symbol, probe orientation, gene description, feature number, location of features and control type of features.