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

Zhang H, Zhu G - Front Microbiol (2015)

Bottom Line: However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures.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).This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University College Station, TX, USA.

ABSTRACT
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. 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). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

No MeSH data available.


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.
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Figure 1: 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.

Mentions: Using qRT-PCR, we have observed rapid intracellular parasite growth (i.e., 34-fold increase of Cp18S rRNA from 3 to 44 hpi) (Figure 1A), which gave us a good dynamic range to quantify the relative parasite growth. For comparison, there was only ~1.6-fold increase of uninfected Hs18S cultured for the same time period (Figure 1B), indicating that parasite grew >20 times faster than the HCT-8 cells in this cultivation system. We also observed that parasite infection slightly reduced the HCT-8 cell growth (i.e., ~10% decrease based on Hs18S rRNA levels) (Figure 1B), which might be attributed by the arrest of host cell growth upon infection and cell death upon the completion of merogony development and the release of parasite merozoites as reported by other investigators (Dobbelaere and Küenzi, 2004; Brunet et al., 2008).


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

Zhang H, Zhu G - Front Microbiol (2015)

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.
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Related In: Results  -  Collection

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Figure 1: 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.
Mentions: Using qRT-PCR, we have observed rapid intracellular parasite growth (i.e., 34-fold increase of Cp18S rRNA from 3 to 44 hpi) (Figure 1A), which gave us a good dynamic range to quantify the relative parasite growth. For comparison, there was only ~1.6-fold increase of uninfected Hs18S cultured for the same time period (Figure 1B), indicating that parasite grew >20 times faster than the HCT-8 cells in this cultivation system. We also observed that parasite infection slightly reduced the HCT-8 cell growth (i.e., ~10% decrease based on Hs18S rRNA levels) (Figure 1B), which might be attributed by the arrest of host cell growth upon infection and cell death upon the completion of merogony development and the release of parasite merozoites as reported by other investigators (Dobbelaere and Küenzi, 2004; Brunet et al., 2008).

Bottom Line: However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures.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).This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University College Station, TX, USA.

ABSTRACT
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. 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). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

No MeSH data available.