Sensitive and Quantitative Measurement of Gene Expression Directly from a Small Amount of Whole Blood

Zheng, Zhi; Luo, Yuling; McMaster, Gary

doi:10.1373/clinchem.2005.065078

Cited by 39 publications

(28 citation statements)

References 35 publications

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“…The next challenge is to identify abnormal levels of mRNA that commonly exist in both normal and disease states. Although normal reference values of these mRNA were quantifi ed using healthy subject population (MAQC Consortium, 2006;Mitsuhashi et al 2006;Peters et al 2007;Zheng et al 2006), the identifi cation of disease-specifi c mRNA levels is one of the current topics in molecular diagnostics development.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

Mitsuhashi

Endo

Obara

et al. 2008

Clinical medicine. Blood disorders

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Apoptosis was induced in heparinized human whole blood by 3 different ways (radiation, bleomycin, or etoposide), and various mRNA were quantifi ed using the method we reported (Clin. Chem. 2006; 52:634-642). We found that cyclin-dependent kinase inhibitor 1A (p21) and p53 upregulated modulator of apoptosis (PUMA) were the most sensitive and universal mRNA markers of apoptosis in leukocytes. In order to defi ne positive and negative responses, a synthetic RNA was spiked into the lysis buffer and the fold increase was calculated. As a result, 837/880 (95.1%) of data points stayed between 0.75 and 1.5 fold increase, and 874/880 (99.3%) were within 0.5-2.0 fold increase. When blood samples from 40 healthy adults were stimulated with 22 different drugs, more than 75% of the samples responded to bleomycin (1 µΜ), idarubicin (2 µΜ), vincristine (1 µΜ), daunorubicin (2 µΜ), cytarabine (10 µΜ), to induce p21 and/or PUMA mRNA, and approximately 25% showed no induction. Signifi cant correlation was found between p21 and PUMA mRNA responses, and between daunorubicin and cytarabine, idarubicin, and vincristine for both p21 and PUMA. The quantifi cation of drug-induced mRNA in whole blood will be considered as ex vivo, and is a suitable platform for biomarker screening as well as a model system for drug sensitivity tests in future.

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Section: Introductionmentioning

confidence: 99%

Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

Mitsuhashi

Endo

Obara

et al. 2008

Clinical medicine. Blood disorders

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“…For each assay, 20 l of fresh or thawed blood or erythrocyte culture of P. falciparum was lysed with 50 l of lysis mixture (Panomics/Affymetrix), 28 l of water, and 2 l of 50-mg/ml proteinase K at 60°C for 1 h with vigorous shaking. The lysates were mixed with probes and hybridized to a capture plate by incubation at 58°C overnight without shaking (25). After the unbound probes were washed off, captured targets were sequentially hybridized, with washing in between, with preamplifier, amplifier, label probe, and substrate as described in the Quantigene assay kit (Panomics/ Affymetrix).…”

Section: Methodsmentioning

confidence: 99%

“…We have sought to develop an easy, sensitive, and reliable molecular assay suitable for large-scale malaria screening by adopting our previously developed sandwich RNA hybridization assay, which uses an enzyme-linked immunosorbent assay (ELISA)-like work flow in a 96-well format without RNA purification, reverse transcription, and target amplification (25). The sandwich hybridization assay is done with a series of oligonucleotide probes containing target-specific sequences and an additional "tail" sequence that is independent of the target sequence but can interact with either the solid support or the detection system.…”

mentioning

confidence: 99%

“…The CEs bind to the capture oligonucleotides conjugated to the surface of each well in 96-well plate and, via cooperative hybridization, capture the associated target RNA. Signal amplification is mediated by DNA amplification molecules that hybridize to the tails of the LEs, and the DNA amplification molecules can then be detected by a chemiluminescent reaction (25).…”

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confidence: 99%

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A Novel, Sensitive Assay for High-Throughput Molecular Detection of Plasmodia for Active Screening of Malaria for Elimination

et al. 2013

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bAlthough malaria remains one of the leading infectious diseases in the world, the decline in malaria transmission in some area makes it possible to consider elimination of the disease. As countries approach elimination, malaria diagnosis needs to change from diagnosing ill patients to actively detecting infections in all carriers, including asymptomatic and low-parasite-load patients. However, few of the current diagnostic methods have both the throughput and the sensitivity required. We adopted a sandwich RNA hybridization assay to detect genus Plasmodium 18S rRNA directly from whole-blood samples from Plasmodium falciparum and Plasmodium vivax patients without RNA isolation. We tested the assay with 202 febrile patients from areas where malaria is endemic, using 20 l of each blood sample in a 96-well plate format with a 2-day enzyme-linked immunosorbent assay (ELISA)-like work flow. The results were compared with diagnoses obtained using microscopy, a rapid diagnostic test (RDT), and genus-specific real-time PCR. Our assay identified all 66 positive samples diagnosed by microscopy, including 49 poorly stored samples that underwent multiple freeze-thaw cycles due to resource limitation. The assay uncovered three falsenegative samples by microscopy and four false-negative samples by RDT and agreed completely with real-time PCR diagnosis. There was no negative sample by our assay that would show a positive result when tested with other methods. The detection limit of our assay for P. falciparum was 0.04 parasite/l. The assay's simple work flow, high throughput, and sensitivity make it suitable for active malaria screening.

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“…Unlike earlier tests of malaria with pooling strategy requiring nucleic acid extraction (17,18 ), CLIP-PCR sacrifices no apparent sensitivity with the pooling of DBS; Cq values remained almost the same with pool sizes of up to 36. This is not surprising, since pooling of negative DBS dose not dilute the concentration of positive ones in the lysate, and during the overnight incubation, the sandwich hybridization retains with high specificity only target RNA on the surface via cooperative hybridization (19,20 ). Nontarget nucleic acids are washed off before formation of the PCR template.…”

Section: Fig 2 Analytical Performance Of Clip-pcrmentioning

confidence: 99%