Monitoring the chromosome 2 intraerythrocytic transcriptome of Plasmodium falciparum using oligonucleotide arrays.

Abstract

Abstract. To test the feasibility of using short oligonucleotide probes to monitor transcript levels in Plasmodium falciparum, a microarray was manufactured containing 4,167 (25 base single-stranded) probes derived from the predicted coding region of P. falciparum chromosome 2. RNA samples from three asexual stages (rings, trophozoites, and schizonts) were labeled and hybridized to the arrays. These results were reproducible, and transcripts were detected for 69% of the 210 genes on chromosome 2. In addition, of the 145 expressed genes, 1/3 appeared to be differentially transcribed during the asexual cycle. Some regions of the chromosome appeared to be transcriptionally silent. Results were confirmed by Northern blot analysis and by quantitative reverse transcriptase-polymerase chain reaction. These data validate the use of relatively short 25-mers for monitoring the expression of a genome that is 82% AT rich.

Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle

“…RT-PCR was performed as previously described (Le Roch et al 2002) using the following primers: 5Ј-GATAATATAAATGT TACTGAACAAGG-3Ј and 5Ј-GGAACACTAATTTCGTTTTGTAC-3Ј (EBA175); 5Ј-ATGGCAAGAACTAAACAAACAGC-3Ј and 5Ј-TTATGATCTTTCTCCACGGATACG-3Ј (histone H3); 5Ј-CAA ACCAATCTGGATCTGCAGAAG-3Ј and 5Ј-CCATCTTGTGCTGA TAATAATTCATC-3Ј (Pfcrt).…”

The Plasmodium falciparum sexual development transcriptome: A microarray analysis using ontology-based pattern identification

“…Total RNA was isolated on each day for 13 days starting on Day 1 for the NF54 parasites and on Days 1-3, 6, 8, and 12 for the 3D7 parasites as previously described [15]. For the purified 3D7 early-stage gametocyte samples, RNA was isolated on Day 1, immediately after CS column separation on Day 2, and for the following 2 days (Days 3 and 4).…”

“…Three micrograms of total RNA, isolated from mixed asexual NF54 parasites and Day 7 NF54 gametocytes, was used for cDNA synthesis using superscript reverse transcriptase and oligo(dT) [12][13][14][15][16][17][18] (Invitrogen, USA). PCR using Extaq polymerase (Takara, Japan) was conducted on 1 l of each cDNA template for 20 cycles using an annealing temperature of 56 • C with primers specific to three hypothetical genes (PF11 0214, PF14 0067, and MAL8P1.16) identified from the microarray data as expressed only during sexual development (PF11 0214: forward primer 5 -GTA ACG AAC AGG AGA TAA ATG GGA TTT GTA-3 , reverse primer 5 -GGG TAT TAC CAT AAC TTT TCA ATT TGT CTC G-3 ; PF14 0067: forward primer 5 -GCA TAT TGT AAA GAA TGG TGT AAA GCC ACG-3 , reverse primer 5 -CCA GCA CTA AAT CCT TCA TGT AAT ACT TTA G-3 ; MAL8P1.16: forward primer 5 -GGT GTT GTA TCA TCT GAA AAT ATT AAG CTC C-3 , reverse primer 5 -CTT AGC CTT TCT TGT TAA ATT CTT CCA AGC-3 ) and the known asexual-stage-specific gene eba-175 (PF07 0128) as an asexual stage positive control (PF07 0128: forward primer 5 -TCA TAG TCA TCA TGG AAA CAG ACA AGA TCG-3 , reverse primer 5 -GTA AAA TAG CTC ATA CAG TAA TCT GAT ACT GC-3 ).…”

k-Means Walk: Unveiling Operational Mechanism of a Popular Clustering Approach for Microarray Data

“…The purpose of microarray is to measure the concentration of mRNA for thousands of genes on samples basis or time series basis, upon which enormous amount of genomic data are usually generated by researchers [3]. An infectious disease with particular reference to malaria is caused by a lethal pathogenic protozoan, Plasmodium falciparum, responsible for major losses and death in Sub-Saharan Africa [4,5]. This disease has attracted intense microarray patronage with large data generation efforts [5][6][7][8][9][10][11][12][13].…”

“…An infectious disease with particular reference to malaria is caused by a lethal pathogenic protozoan, Plasmodium falciparum, responsible for major losses and death in Sub-Saharan Africa [4,5]. This disease has attracted intense microarray patronage with large data generation efforts [5][6][7][8][9][10][11][12][13]. In addition, from the effort of Kissinger et al [14] and Bahl et al [15], Plasmodium spp data are now accumulated and integrated into a database called PlasmoDB.…”