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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.


Related in: MedlinePlus

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 CT 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.
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Figure 2: 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 CT 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.

Mentions: We observed that heat treatment of cell lysates at 75°C for 10–30 min significantly improved the release of parasite RNA. When samples were lysed in l00, 150, or 200 μL/well lysis buffer, heat treatment for 10, 20, or 30 min reduced the CT[Cp18S] values from 24.4, 24.0, and 23.9 to 22.4, 22.3, and 22.2 in the 100 μL/well group, from 25.3, 24.9, and 24.5 to 23.6, 23.4, and 23.3 in the 150 μL/well group, or from 25.6, 25.5, and 25.2 to 23.9, 23.6, and 23.6 in the 200 μL/well group, respectively (p < 0.005 by Student t-test in all samples in comparison to the unheated counterparts) (Figure 2). However, we also observed certain levels of heat-induced host cell RNA degradation (i.e., CT[Hs18S] values in heated samples were increased by ~0.2–1.0) (Figure 2). One possible explanation was that the host cell membranes could be rapidly lysed to release RNA with ice-cold lysis buffer (as heating was not included in the manufacturer's protocol), and there was a small window of time at the beginning of the heating process for the host cell RNases to become active before being deactivated by heat. On the other hand, more parasite RNA was released after the temperature reached to 75°C and RNases were deactivated. However, the final host cell RNA concentrations appeared to be consistent within each experimental group (i.e., CV values at 0.75–3.04% for all groups), indicating that the RNA degradation would not result in inconsistency in subsequent determination of relative Hs18S levels. Finally, based on the observation that heating between 10 and 20 min in wells containing 200 μL lysis buffer resulted in the least degradation of host cell RNA (ΔCT at 0.20–0.25), but the most improvement in releasing parasite RNA (ΔCT at −1.8 to −2.0), we determined that the optimal condition was the use of 200 μL iScript™ RT-qPCR Sample Preparation Reagent and heating for 15 min.


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)

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 CT 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: 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 CT 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.
Mentions: We observed that heat treatment of cell lysates at 75°C for 10–30 min significantly improved the release of parasite RNA. When samples were lysed in l00, 150, or 200 μL/well lysis buffer, heat treatment for 10, 20, or 30 min reduced the CT[Cp18S] values from 24.4, 24.0, and 23.9 to 22.4, 22.3, and 22.2 in the 100 μL/well group, from 25.3, 24.9, and 24.5 to 23.6, 23.4, and 23.3 in the 150 μL/well group, or from 25.6, 25.5, and 25.2 to 23.9, 23.6, and 23.6 in the 200 μL/well group, respectively (p < 0.005 by Student t-test in all samples in comparison to the unheated counterparts) (Figure 2). However, we also observed certain levels of heat-induced host cell RNA degradation (i.e., CT[Hs18S] values in heated samples were increased by ~0.2–1.0) (Figure 2). One possible explanation was that the host cell membranes could be rapidly lysed to release RNA with ice-cold lysis buffer (as heating was not included in the manufacturer's protocol), and there was a small window of time at the beginning of the heating process for the host cell RNases to become active before being deactivated by heat. On the other hand, more parasite RNA was released after the temperature reached to 75°C and RNases were deactivated. However, the final host cell RNA concentrations appeared to be consistent within each experimental group (i.e., CV values at 0.75–3.04% for all groups), indicating that the RNA degradation would not result in inconsistency in subsequent determination of relative Hs18S levels. Finally, based on the observation that heating between 10 and 20 min in wells containing 200 μL lysis buffer resulted in the least degradation of host cell RNA (ΔCT at 0.20–0.25), but the most improvement in releasing parasite RNA (ΔCT at −1.8 to −2.0), we determined that the optimal condition was the use of 200 μL iScript™ RT-qPCR Sample Preparation Reagent and heating for 15 min.

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.


Related in: MedlinePlus