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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.


Evaluation of drug efficacy using the new qRT-PCR assay. (A) Standard curves showing the relationship between the number of inoculated oocysts and ΔCT(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.
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Figure 4: Evaluation of drug efficacy using the new qRT-PCR assay. (A) Standard curves showing the relationship between the number of inoculated oocysts and ΔCT(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.

Mentions: We also generated standard curves from cells infected with various numbers of parasites. We considered that this approach was better than the traditional method using serially diluted RNA templates, because standard curves derived from various numbers of infected parasites would correct systematic errors from infection, sample preparation to qRT-PCR procedures, rather than to only correct the errors introduced at the qRT-PCR step. In this assay, linear relationship was observed between the ΔCT values and the samples isolated from cells infected with 64–4 × 104 oocysts per well (Figure 4A). The ΔCT values from samples infected with more or less parasites frequently fell outside of the linear range (data not shown). The linear dynamic range for this new assay (~156-fold) was smaller than our previously reported assay using conventional protocols (Cai et al., 2005). There were two possible explanations. Firstly, the 96-well format had lower limits on the number of parasites that could be cultured in 96-well without density effect. Secondly, in comparison to conventional RNA isolation methods, cell lysates contained much less RNA due to the less efficiency in releasing RNA by the lysis buffer and higher dilutions needed to minimize the inhibition of RT-PCR reactions caused by substances present in the lysis buffer. However, the 156-fold linear dynamic range was sufficient for testing drug efficacy in vitro, as it could theoretically resolve < 1% changes of parasite loads in the assay system.


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)

Evaluation of drug efficacy using the new qRT-PCR assay. (A) Standard curves showing the relationship between the number of inoculated oocysts and ΔCT(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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585199&req=5

Figure 4: Evaluation of drug efficacy using the new qRT-PCR assay. (A) Standard curves showing the relationship between the number of inoculated oocysts and ΔCT(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.
Mentions: We also generated standard curves from cells infected with various numbers of parasites. We considered that this approach was better than the traditional method using serially diluted RNA templates, because standard curves derived from various numbers of infected parasites would correct systematic errors from infection, sample preparation to qRT-PCR procedures, rather than to only correct the errors introduced at the qRT-PCR step. In this assay, linear relationship was observed between the ΔCT values and the samples isolated from cells infected with 64–4 × 104 oocysts per well (Figure 4A). The ΔCT values from samples infected with more or less parasites frequently fell outside of the linear range (data not shown). The linear dynamic range for this new assay (~156-fold) was smaller than our previously reported assay using conventional protocols (Cai et al., 2005). There were two possible explanations. Firstly, the 96-well format had lower limits on the number of parasites that could be cultured in 96-well without density effect. Secondly, in comparison to conventional RNA isolation methods, cell lysates contained much less RNA due to the less efficiency in releasing RNA by the lysis buffer and higher dilutions needed to minimize the inhibition of RT-PCR reactions caused by substances present in the lysis buffer. However, the 156-fold linear dynamic range was sufficient for testing drug efficacy in vitro, as it could theoretically resolve < 1% changes of parasite loads in the assay system.

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.