Journal: Frontiers in Microbiology

Article Title: Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

doi: 10.3389/fmicb.2015.00991

Figure Lengend Snippet: Comparison of the growths between Cryptosporidium parvum and host cells (HCT-8 cell line) cultured in 96-well plates for 44 h post-infection (hpi) as determined by detecting their relative levels of 18S rRNA transcripts . (A) The growth rate of C. parvum in HCT-8 cells between 3 and 44 hpi. The levels of 18S rRNA transcripts from the parasite (Cp18S) and host cells (Hs18S) were determined by qRT-PCR, and those of Hs18S were used for normalization before calculating the fold changes in Cp18S transcripts. (B) The relative levels of Hs18S in HCT-8 cells infected with C. parvum for 3 and 44 hpi. Uninfected cells were grown in parallel under the same condition for the same time periods. Bars represent standard error of the mean (SEM, n = 9) from three independent experiments. (C) Micrographs showing the development of C. parvum cultured in HCT-8 cells at 3 and 44 h post-infection (hpi) time points, in which intracellular parasites were labeled with a rabbit antiserum against C. parvum sporozoite total membrane proteins and a TRITC-conjugated goat anti-rabbit IgG secondary antibody and counter-stained with DAPI for nuclei. DIC, differential inference contrast microscopy. DAPI, 4′, 6-diamidino-2-phenylindole for counterstaining of nuclei. TRITC, Tetramethylrhodamine.

Article Snippet: Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).

Techniques: Cell Culture, Infection, Quantitative RT-PCR, Labeling, Staining, Microscopy

Journal: Frontiers in Microbiology

Article Title: Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

doi: 10.3389/fmicb.2015.00991

Figure Lengend Snippet: Effects of the amount of lysis buffer and heat treatment on the release of RNA from Cryptosporidium parvum and HCT-8 cells . Cell lysates were first diluted by 100 and 2000 times with nuclease-free water prior to qRT-PCR detection for Cp18S and Hs18S rRNA transcripts, respectively. The plotted C T values were not calibrated to equal volume of lysis buffer. Bars represent standard error of the mean (SEM, n = 6). Heat treatment vs. un-treatment control, p < 0.005 by Student's t -test in all samples.

Article Snippet: Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).

Techniques: Lysis, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

doi: 10.3389/fmicb.2015.00991

Figure Lengend Snippet: Specific detection of Cp18S and Hs18S rRNA transcripts as demonstrated by amplification curve (A) and melting curve (B) analyses of a representative plate . Lysates of HCT-8 cells grown in 96-well plates and infected with Cryptosporidium parvum for 44 h were used in qRT-PCR reactions in 384-well format.

Article Snippet: Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).

Techniques: Amplification, Infection, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

doi: 10.3389/fmicb.2015.00991

Figure Lengend Snippet: Summary of the C T values in the uniformity assay using the simplified qRT-PCR assay ^a .

Article Snippet: Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

doi: 10.3389/fmicb.2015.00991

Figure Lengend Snippet: Assay validation on the uniformity of the simplified qRT-PCR assay based on normalized ΔC T values ^a .

Article Snippet: Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

doi: 10.3389/fmicb.2015.00991

Figure Lengend Snippet: Evaluation of drug efficacy using the new qRT-PCR assay . (A) Standard curves showing the relationship between the number of inoculated oocysts and ΔC T(Cp18S−Hs18S) values; (B) Dose-response curve on the in vitro anti-cryptosporidial activity of paromomycin. Bars represent standard errors of the mean (SEM) derived from at least two biological replicates.

Article Snippet: Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).

Techniques: Quantitative RT-PCR, In Vitro, Activity Assay, Derivative Assay